Elicit: CDK4/6 Inhibition in HR+ HER2- Breast Cancer

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CDK4/6 Inhibition in HR+ HER2- Breast Cancer

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May 5, 2026

What are the downstream signaling changes and biomarkers of CDK4/6 inhibition in HR+ HER2- breast cancer?

CDK4/6 inhibition induces cell cycle arrest through Rb pathway suppression and E2F downregulation, reduces immunosuppressive cells, and alters growth factor signaling, with key biomarkers including high ER and intact Rb for sensitivity versus IFN pathway activation, Cyclin D1/CDK4 overexpression, and PI3K/mTOR hyperactivation for resistance.

Abstract

CDK4/6 inhibition in HR+ HER2- breast cancer induces multiple downstream signaling changes beyond primary cell cycle arrest. Prevention of Rb phosphorylation leads to G1 arrest and downregulation of E2F-regulated genes including RRM2, TOPO2A, MKI67, MCM7, and CDK2, with treatment shifting tumors from high-risk luminal B to low-risk luminal A molecular phenotype. Growth factor signaling pathways show context-dependent alterations: baseline FGFR2 and ERBB3 expression associate with greater benefit from CDK4/6 inhibition, while acquired PI3K/mTOR hyperactivation drives resistance through Cyclin D1 and CDK4 overexpression. Immune signaling exhibits dual patterns, with beneficial reduction of immunosuppressive Tregs and MDSCs during treatment (p<0.0001) but aberrant IFN/STAT1 pathway activation associated with intrinsic and acquired resistance. Additional changes include suppression of ubiquitin-conjugating enzymes UBE2C, UBE2S, and UBE2T, downregulation of DNA repair pathways in resistant cells, and impairment of radiation-induced ERK and NF-κB/c-Myc signaling.

Predictive biomarkers of resistance include the IFN-related palbociclib-resistance signature (IRPS), overexpressed Cyclin D1 and CDK4 proteins, IL6/STAT3 pathway activation, and baseline Treg levels, while markers of sensitivity include high ER levels, Rb wild-type status, low cyclin E, and low p16. Despite comprehensive biomarker analysis in 666 patients, the PALOMA-2 trial found no single baseline marker predicting lack of benefit, suggesting broad initial efficacy. However, approximately 25-35% of patients demonstrate intrinsic resistance, and nearly all eventually acquire resistance through mechanisms including RB1 loss, cyclin E1 amplification, ERBB2/BRAF mutations, and PI3K/mTOR pathway activation. Strategies to overcome resistance include PI3K/mTOR inhibition to restore CDK4/6 inhibitor sensitivity and combined STAT3/PARP inhibition, with mTOR pathway activation correlating with clinical benefit to triplet therapy in CDK4/6 inhibitor-pretreated patients.

Methods

We analyzed 10 sources from an initial pool of 200, using 7 screening criteria. Each paper was reviewed for 7 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Population - Cancer Type and Subtype, Intervention - CDK4/6 Inhibitors, Outcomes - Mechanistic Data, Outcome Level - Molecular Focus, Population Specificity, Intervention Isolation, Publication Type

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “What are the downstream signaling changes and biomarkers of CDK4/6 inhibition in HR+ HER2- breast cancer?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract study design and population characteristics for CDK4/6 inhibition research in HR+ HER2- breast cancer, including:

Extract details about CDK4/6 inhibitor treatment in HR+ HER2- breast cancer, including:

Extract all downstream signaling pathway changes and molecular alterations following CDK4/6 inhibition in HR+ HER2- breast cancer, including:

Extract all biomarker results related to CDK4/6 inhibition in HR+ HER2- breast cancer, including:

Extract specific mechanisms of resistance to CDK4/6 inhibitors in HR+ HER2- breast cancer, including:

Extract how molecular findings relate to clinical outcomes in CDK4/6 inhibitor-treated HR+ HER2- breast cancer patients, including:

Extract methodological details for molecular analyses in CDK4/6 inhibitor studies in HR+ HER2- breast cancer, including:

Results

Characteristics of Included Studies

The review included 10 studies investigating downstream signaling changes and biomarkers of CDK4/6 inhibition in HR+ HER2- breast cancer, comprising 3 clinical studies and 7 preclinical studies.

Study

Full text retrieved?

Study Type

Sample Size/Population

Disease Setting

CDK4/6 Inhibitor Used

R. Finn et al., 2019

Yes

Randomized, placebo-controlled, phase III clinical trial

666 postmenopausal women with HR+/HER2- metastatic breast cancer

Metastatic, treatment-naive

Palbociclib 125 mg daily (3 weeks on, 1 week off) plus letrozole

Carmine De Angelis et al., 2021

No

Preclinical

Cell lines (MCF7, T47D)

Not applicable

Palbociclib

Neil A. O’Brien et al., 2018

Yes

Preclinical

44 breast cancer cell lines

Not applicable

Abemaciclib (continuous daily, 50 mg/kg)

Zijie Cai et al., 2022

No

Preclinical

Palbociclib-resistant cell lines

Advanced

Palbociclib

Chih-Yi Lin et al., 2022

No

Preclinical

MCF7 and T47D cell lines

Not applicable

Palbociclib, ribociclib, abemaciclib

N. Kettner et al., 2019

No

Preclinical

MCF-7 and T47D cells

Not mentioned

Palbociclib

J. G. T. Zañudo et al., 2022

Yes

Phase I/II clinical trial

32 female patients, median age 55.5 years

Metastatic HR+ HER2-, pretreated with prior CDK4/6 inhibitors

Palbociclib 100 mg plus everolimus 5 mg plus exemestane 25 mg

E. Knudsen & A. Witkiewicz, 2016

Yes

Preclinical

MCF7 and T47D cell lines, MDA-MB-231 xenograft

Not explicitly stated

Palbociclib (in vitro: 100 nM-1 µM; in vivo: 125 mg/kg)

F. Scirocchi et al., 2022

Yes

Prospective observational study

50 consecutive women, median age 62 years

Metastatic HR+/HER2-, mostly first-line treatment

Palbociclib, ribociclib, abemaciclib with hormone therapy

Wen-Chi Yang et al., 2024

No

Not mentioned

Not mentioned

Not mentioned

Not specified

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The studies investigated different aspects of CDK4/6 inhibition, including clinical efficacy, resistance mechanisms, downstream signaling pathways, and immune modulation. Three studies used palbociclib exclusively, one used abemaciclib, three tested multiple CDK4/6 inhibitors, and two did not specify the inhibitor. Among clinical studies, one was treatment-naive and one enrolled CDK4/6 inhibitor-pretreated patients.

Downstream Signaling Changes Following CDK4/6 Inhibition

Cell Cycle Pathway Changes

CDK4/6 inhibition consistently affected core cell cycle machinery across studies. The primary mechanism involved prevention of retinoblastoma (Rb) protein hyperphosphorylation, leading to G1 cell cycle arrest. This was accompanied by reduction in cell cycle progression markers including phospho-Rb, TOPOIIa, phosphohistone-H3, and FOXM1. Transcriptional analysis revealed downregulation of E2F-regulated genes including RRM2, TOPO2A, MKI67, MCM7, and CDK2, which tracked with growth inhibitory response.

CDK4/6 inhibition induced a molecular shift from high-risk luminal B to low-risk luminal A phenotype, with genes repressed by treatment strongly associated with clinical prognosis in ER+/HER2- cases. Importantly, treatment elicited induction of a comparable number of genes involved in multiple processes beyond cell cycle suppression, suggesting complex transcriptional rewiring.

In resistance settings, alterations in cell cycle machinery included loss-of-function alterations in RB1, high cyclin E1 mRNA expression, amplifications of AURKA, and CDK6 overexpression. Palbociclib-resistant cells exhibited overexpression of Cyclin D1 and CDK4 proteins due to upregulated protein synthesis, with silencing of these proteins leading to cell cycle arrest.

Growth Factor Signaling Alterations

Multiple growth factor signaling pathways showed altered activity following CDK4/6 inhibition. Tumors with increased expression of FGFR2 and ERBB3 mRNA demonstrated greater PFS gain from palbociclib addition, suggesting interplay between steroid hormone and peptide growth factor signaling drives CDK4/6 dependence.

In resistant cells, the PI3K/mTOR pathway exhibited hyper-activation, leading to increased phosphorylation of 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Tumor samples after palbociclib progression showed significantly higher levels of Cyclin D1, CDK4, p-AKT, and p-4E-BP1. Genomic analysis revealed oncogenic pathways including PI3K/mTOR, FGFR, and ERBB were enriched in resistant samples, with consistency between oncogenic mutations and transcriptional signature activities in ER, PI3K/AKT/mTOR, and RTK/MAPK pathways.

CDK4/6 inhibition also reduced radiation-induced activation of ERK and NF-κB/c-Myc signaling pathways, suggesting broader effects on growth factor-activated cascades.

Immune Signaling Changes

Aberrant interferon (IFN) signaling emerged as a key resistance mechanism. High IFN signaling was associated with reduced CDK4/6 inhibitor sensitivity, with an “IFN-related palbociclib-resistance Signature” (IRPS) derived from transcriptomic analysis. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared to untreated counterparts. The IRPS score was significantly higher in luminal B versus luminal A subtypes and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.

In palbociclib-resistant cells, the IL6/STAT3 pathway was induced while DNA repair and estrogen receptor pathways were downregulated. Patient samples post-palbociclib progression showed altered IL6/STAT3 signaling compared to pretreatment samples.

CDK4/6 inhibition treatment significantly downregulated circulating regulatory T cells (Tregs) and myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) from baseline (p<0.0001 and p<0.05, respectively). The effector Treg subset (CD4+CD25+FOXP3highCD45RA-) was strongly reduced (p<0.0001). Conversely, treatment increased levels of CD4+ T cells and anti-tumor CD137+CD8+ T cells (p<0.05), suggesting relief from immunosuppression.

Lower PD-1 levels were associated with greater benefit from palbociclib plus letrozole, while the PD-1 signaling pathway was associated with reduced PFS benefit.

Ubiquitin-Proteasome Pathway Modulation

CDK4/6 inhibitors suppressed expression of three ubiquitin-conjugating enzymes: UBE2C, UBE2S, and UBE2T. Palbociclib and ribociclib decreased UBE2C at both mRNA and protein levels, though this phenomenon was not shared with abemaciclib. These E2 enzymes modulate several E3 ubiquitin ligases, including the APC/C complex which plays a role in G1/S progression.

DNA Repair and Additional Pathways

CDK4/6 inhibitors impaired the DNA-DSB repair mechanism activated by radiation. In palbociclib-resistant cells, DNA repair pathways were downregulated, and estrogen receptor pathways showed reduced activity. Patient samples post-progression demonstrated alterations in estrogen receptor, DNA repair, and IL6/STAT3 signaling compared to pretreatment.

Metabolic pathways were also affected, with increased glucose and glutamine metabolism, increased cell size, and elevated ATP levels observed following CDK4/6 inhibition. However, these metabolic changes were mitigated by concurrent endocrine therapy.

Biomarker Findings

Predictive Biomarkers of Response and Resistance

Multiple baseline markers showed associations with CDK4/6 inhibitor response. Higher CDK4 levels were associated with endocrine resistance, which was mitigated by palbociclib addition. Lower PD-1 levels at baseline associated with greater benefit from palbociclib plus letrozole.

For abemaciclib, predictive biomarkers of response included high ER levels, Rb wild-type status, high Rb total and phosphoprotein levels, lack of cyclin E amplification, low cyclin E protein, and low p16 protein levels. These markers reflected dependency on the cyclinD:CDK4/6:Rb signaling pathway.

Resistance-associated predictive biomarkers included overexpressed Cyclin D1 and CDK4 proteins, the IFN-related palbociclib-resistance Signature (IRPS), IL6/STAT3 pathway induction, and downregulation of DNA repair and estrogen receptor pathways. PI3K/AKT/mTOR mutations and high mTORC1 pathway activity correlated with clinical benefit to combined estrogen receptor, CDK4/6, and mTOR inhibition.

In the clinical trial testing triplet therapy after CDK4/6 inhibitor progression, genomic and transcriptomic features enabled identification of known or putative drivers of resistance in nearly every patient (22/23), with several patients showing transcriptomic features as sole drivers.

Prognostic Biomarkers

Higher ESR1 gene expression was associated with greater benefit from both placebo plus letrozole and palbociclib plus letrozole. Genes repressed by CDK4/6 inhibition were strongly associated with improved prognosis and reduced risk of recurrence.

The IRPS score correlated with poor prognosis in primary ER+/HER2- tumors. UBE2C/UBE2T expression levels were associated with breast cancer survival. Activating ESR1 mutations were identified in Luminal A/B subtypes while ERBB2/BRAF mutations occurred in HER2-E subtypes in mutually exclusive patterns.

Baseline Treg levels were associated with response to CDK4/6 inhibitor treatment, with Treg levels both at baseline and during treatment showing prognostic value.

Pharmacodynamic Biomarkers

CDK4 levels showed association with endocrine resistance, suggesting a role in drug activity. The pharmacodynamic biomarkers for abemaciclib included mRNA expression of RRM2, TOPO2A, MKI67, MCM7, and CDK2, which are directly regulated by E2F transcription factor downstream of Rb.

Hyper-activated PI3K/mTOR pathway markers, including p-AKT and p-4E-BP1, served as pharmacodynamic indicators. Decrease in UBE2C at mRNA and protein levels by palbociclib and ribociclib demonstrated drug activity on the ubiquitin-proteasome pathway. Activation of IFN/STAT1 signaling in resistant derivatives indicated pathway engagement.

Reduction in Tregs and MDSCs alongside increase in CD4+ and CD137+CD8+ T cells indicated immune modulation by CDK4/6 inhibitors. Impaired DNA-DSB repair mechanism and reduced ERK and NF-κB/c-Myc signaling pathways also served as pharmacodynamic markers.

Resistance Mechanisms

Intrinsic Resistance

Approximately 25-35% of patients demonstrated intrinsic resistance to CDK4/6 inhibitors. Molecular features of intrinsic resistance included high IFN signaling and the IRPS signature. High p16 protein levels and amplification of cyclin E were associated with reduced sensitivity to abemaciclib.

Baseline loss-of-function alterations in RB1, high cyclin E1 expression, low ER expression, and basal molecular subtype represented intrinsic resistance mechanisms. Higher CDK4 levels at baseline were associated with endocrine resistance, though this could be mitigated by palbociclib.

Acquired Resistance

Nearly all patients eventually acquired resistance during CDK4/6 inhibitor treatment. Acquired resistance mechanisms included activation of IFN/STAT1 signaling, dramatic upregulation compared to untreated cells. IL6/STAT3 pathway induction occurred in palbociclib-resistant cells, along with downregulation of DNA repair and estrogen receptor pathways.

Acquired genomic alterations driving resistance included activating mutations in ERBB2, BRAF, and PIK3CA, and amplifications in FGFR1 and ERBB2. Overexpression of Cyclin D1 and CDK4 proteins developed in resistant cells due to hyper-activation of the PI3K/mTOR pathway.

Dysregulation of key signaling pathways downstream of ER and/or HER2 alterations contributed to acquired resistance, particularly in metastatic settings. Aberrant mitogenic signaling pathway activation represented another acquired resistance mechanism.

Cross-Resistance Patterns

Palbociclib-resistant cells demonstrated cross-resistance to other CDK4/6 inhibitors and endocrine therapy. This included resistance to estrogen receptor downregulation. The IRPS signature correlated with increased gene expression of immune checkpoints and endocrine resistance. Patients showing resistance to CDK4/6 inhibitors also demonstrated resistance to endocrine therapy.

Strategies to Overcome Resistance

Targeting the PI3K/mTOR pathway with specific PI3Kα inhibitor (BYL719) or mTOR inhibitor (everolimus) reduced Cyclin D1 and CDK4 protein levels and restored sensitivity to palbociclib. Combined inhibition of STAT3 and PARP significantly increased cell death in palbociclib-resistant cells, suggesting this combination could effectively treat acquired resistance.

Targeting the mTOR pathway showed promise, as mTOR pathway activation correlated with clinical benefit. Selection of patients based on intact Rb pathway signaling could identify those who may benefit from abemaciclib therapy. Targeting the interplay between steroid hormone and peptide growth factor signaling was proposed to overcome resistance.

Endocrine therapy prevented compensatory growth that could contribute to resistance, and discontinuous dosing schedules may limit mitogenic signaling to prevent early resistance. Using CDK4/6 inhibitors to reduce immunosuppression and enhance checkpoint inhibitors represented another strategy.

Clinical Correlations of Molecular Findings

Response Rates and Molecular Subgroups

The PALOMA-2 trial demonstrated that palbociclib plus letrozole conferred efficacy on both luminal A and B patients, with no single biomarker or cassette of markers associated with lack of benefit from combination treatment.

In the CDK4/6 inhibitor-pretreated population, the clinical benefit rate for triplet therapy (exemestane plus everolimus plus palbociclib) was 18.8%. Among patients treated with CDK4/6 inhibitors plus aromatase inhibitors in first-line, 59% achieved complete or partial response, while in second-line with fulvestrant, 14% achieved response.

The IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6 inhibitors. Sensitivity to abemaciclib was observed predominantly in luminal ER+/HER2- and ER+/HER2+ subtypes.

Progression-Free Survival Associations

Higher CDK4 levels were associated with endocrine resistance and shorter PFS in the placebo arm, with this resistance mitigated by palbociclib. Lower PD-1 levels associated with greater PFS benefit from palbociclib plus letrozole. Active growth factor signaling, exemplified by FGFR2 and ERBB3 expression, was associated with greater PFS gain from palbociclib addition.

The IRPS score correlated with increased gene expression of immune checkpoints and poor prognosis, suggesting associations with shortened PFS. Improved PFS was associated with abemaciclib plus fulvestrant in the MONARCH-2 clinical trial.

Resistance to CDK4/6 inhibitors was associated with estrogen receptor downregulation and alterations in IL6/STAT3 and DNA damage response pathways. The hyperactivation of the PI3K/mTOR pathway in resistant cells suggested associations with shortened PFS.

CDK4/6 inhibition treatment shifted ER+/HER2- models from high-risk luminal B to low-risk luminal A molecular phenotype, associated with improved prognosis.

Immune Correlates of Response

The decrease in Treg levels was significantly greater in responder patients compared to non-responder patients. Treg levels at baseline (>T0) and Treg effector levels at T0 were significant prognostic factors, with a multivariate analysis model correctly classifying 74% of patients. The area under the ROC curve was 0.701, indicating moderate predictive accuracy for immune biomarkers.

Synthesis

The studies revealed substantial mechanistic heterogeneity in CDK4/6 inhibitor response and resistance, requiring synthesis across multiple dimensions to explain divergent findings.

Context-Dependent Mechanisms

Different resistance mechanisms dominated in distinct clinical contexts. Intrinsic resistance (25-35% of patients) was primarily characterized by baseline pathway alterations including high IFN signaling, RB1 loss, high cyclin E1, and immunosuppressive phenotypes. In contrast, acquired resistance that developed during treatment involved activation of compensatory signaling pathways not present at baseline, including IL6/STAT3 pathway induction, PI3K/mTOR hyperactivation, and ERBB2/BRAF mutations.

This temporal distinction explains why biomarker studies using baseline samples (PALOMA-2) found no single marker predicting lack of benefit, while studies examining progression samples identified multiple resistance drivers. Both observations are correct within their respective timeframes: CDK4/6 inhibitors provide broad initial benefit regardless of baseline markers, but acquired alterations subsequently drive progression.

The setting of prior CDK4/6 inhibitor exposure further modified resistance patterns. In the triplet therapy trial enrolling CDK4/6 inhibitor-pretreated patients, resistance mechanisms were identifiable in nearly all patients (22/23), with genomic drivers in RTK/MAPK, PI3K/AKT/mTOR pathways prevalent. The modest 18.8% clinical benefit rate in this pretreated population contrasted with the broad efficacy in treatment-naive patients, consistent with accumulated resistance alterations limiting subsequent treatment options.

Integration of Transcriptomic and Genomic Features

Studies employing both genomic and transcriptomic analyses revealed that genomic resistance mechanisms associated with corresponding transcriptomic signatures. ESR1 mutations linked with high estrogen receptor pathway activity in Luminal A/B subtypes, while ERBB2/BRAF mutations associated with high RTK/MAPK pathway activity in HER2-E subtypes. This concordance validated transcriptomics as complementary to genomics, with transcriptomic features identifying pathway drivers even absent genomic alterations.

The molecular shift from luminal B to luminal A phenotype following CDK4/6 inhibition provided mechanistic insight into clinical benefit. Genes repressed by CDK4/6 inhibition strongly associated with poor prognosis, explaining why their suppression improved outcomes. However, induced genes also associated with improved outcomes, suggesting dual beneficial effects beyond simple cell cycle arrest. The induction of growth-promoting pathways that could contribute to resistance was antagonized by concurrent endocrine therapy, explaining the superior efficacy of combination versus monotherapy.

Pathway Crosstalk and Therapeutic Implications

Growth factor and hormone signaling exhibited complex interdependence. Tumors with active FGFR2/ERBB3 signaling showed greater CDK4/6 inhibitor benefit, suggesting growth factor pathway activation increased CDK4/6 dependence rather than conferring resistance. Conversely, PI3K/mTOR hyperactivation in resistant cells drove Cyclin D1/CDK4 overexpression, creating CDK4/6 inhibitor insensitivity through target protein excess. This distinction explained why PI3K/mTOR pathway markers showed opposing relationships with benefit depending on context: moderate baseline activity associated with CDK4/6 inhibitor sensitivity, while acquired hyperactivation drove resistance.

The differential effects of palbociclib, ribociclib, and abemaciclib on UBE2C expression indicated mechanistic distinctions between CDK4/6 inhibitors despite shared primary targets. This heterogeneity may contribute to non-cross-resistance and sequential efficacy, though clinical validation remains limited.

Immune Modulation as Dual Mechanism

CDK4/6 inhibition’s immune effects showed apparent contradictions: treatment reduced immunosuppressive Tregs and MDSCs with benefit correlating with immune activation, yet high baseline IFN signaling associated with resistance. This paradox resolves through mechanistic separation: CDK4/6 inhibitors beneficially reduced regulatory immune populations suppressing anti-tumor immunity, while aberrant chronic IFN pathway activation represented a resistance mechanism independent of CDK4/6 inhibitor-induced immune changes. The IRPS signature’s enrichment in luminal B versus luminal A tumors and correlation with poor prognosis indicated this represented a tumor-intrinsic resistance mechanism rather than response to treatment.

Methodological Heterogeneity and Quality Weighting

Preclinical studies (7/10 sources) provided detailed mechanistic insights into signaling changes and resistance pathways but used cell lines and xenografts that may not fully recapitulate clinical heterogeneity. The phase III PALOMA-2 trial with 666 patients represented the highest-quality evidence for baseline biomarker associations, finding remarkably consistent benefit across subgroups. However, its baseline-only design limited detection of acquired resistance mechanisms identified in studies with serial sampling.

The phase I/II triplet therapy trial employed comprehensive genomic and transcriptomic profiling at progression, providing high-quality evidence for acquired resistance mechanisms despite modest sample size (32 patients). The prospective observational study with 50 patients contributed unique evidence on immune dynamics through serial blood sampling, though lacked genomic correlates. Studies with abstract-only availability provided valuable mechanistic hypotheses requiring validation.

Resistance mechanisms with convergent evidence across clinical and preclinical studies merit greatest confidence: PI3K/mTOR pathway activation was identified in both palbociclib-resistant cell lines and patient tumor samples, with clinical correlation to treatment benefit when targeted. Similarly, immune checkpoint activation was observed in both IFN-high cell lines and patient samples. Mechanisms identified solely in preclinical models (e.g., UBE2C suppression) require clinical validation.

References

R. Finn, Yuan Liu, Zhou Zhu, Miguel Martín, H. Rugo, and 8 more\ (2019).Biomarker Analyses of Response to Cyclin-Dependent Kinase 4/6 Inhibition and Endocrine Therapy in Women with Treatment-Naïve Metastatic Breast Cancer. Clinical Cancer Research

E. Knudsen, A. Witkiewicz\ (2016).Defining the transcriptional and biological response to CDK4/6 inhibition in relation to ER+/HER2- breast cancer. OncoTarget

F. Scirocchi, S. Scagnoli, A. Botticelli, A. Di Filippo, C. Napoletano, and 7 more\ (2022).Immune effects of CDK4/6 inhibitors in patients with HR+/HER2− metastatic breast cancer: Relief from immunosuppression is associated with clinical response. EBioMedicine

Wen-Chi Yang, M. Wei, Yi-Hsuan Lee, Chiun-Sheng Huang, S. Kuo\ (2024).Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway. Translational Oncology

Carmine De Angelis, Xiaoyong Fu, M. Cataldo, A. Nardone, Resel Pereira, and 25 more\ (2021).Activation of the IFN Signaling Pathway is Associated with Resistance to CDK4/6 Inhibitors and Immune Checkpoint Activation in ER-Positive Breast Cancer. Clinical Cancer Research

Neil A. O’Brien, D. Conklin, R. Beckmann, Tong Luo, Kevin Chau, and 8 more\ (2018).Preclinical Activity of Abemaciclib Alone or in Combination with Antimitotic and Targeted Therapies in Breast Cancer. Molecular Cancer Therapeutics

Zijie Cai, Jingru Wang, Yudong Li, Qianfeng Shi, Liang Jin, and 12 more\ (2022).Overexpressed Cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors. Science China Life Sciences

Chih-Yi Lin, Chung-Jen Yu, Chun-Yu Liu, T. Chao, Chi-Cheng Huang, and 2 more\ (2022).CDK4/6 inhibitors downregulate the ubiquitin-conjugating enzymes UBE2C/S/T involved in the ubiquitin–proteasome pathway in ER + breast cancer. Clinical and Translational Oncology

N. Kettner, S. Vijayaraghavan, M. Durak, Tuyen N Bui, Mehrnoosh Kohansal, and 17 more\ (2019).Combined Inhibition of STAT3 and DNA Repair in Palbociclib-Resistant ER-Positive Breast Cancer. Clinical Cancer Research

J. G. T. Zañudo, R. Barroso-Sousa, Esha Jain, Qing Jin, Tianyu Li, and 14 more\ (2022).Genomic and Transcriptomic Determinants of Resistance to CDK4/6 Inhibitors and Response to Combined Exemestane plus Everolimus and Palbociclib in Patients with Metastatic Hormone Receptor Positive Breast Cancer. medRxiv

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Biomarker Analyses of Response to Cyclin-Dependent Kinase 4/6 Inhibition and Endocrine Therapy in Women with Treatment-Naïve Metastatic Breast Cancer

R. Finn, Yuan Liu, Zhou Zhu, Miguel Martín, H. Rugo, V. Diéras, S. Im, K. Gelmon, N. Harbeck, D. Lu, E. Gauthier, Cynthia Huang Bartlett, D. Slamon

Clinical Cancer Research·

2019·

146 citations

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Study Design

- Study type: Randomized, placebo-controlled, phase III clinical trial - Sample size and patient characteristics: 666 postmenopausal women with HR+/HER2- metastatic breast cancer - Disease setting: Metastatic breast cancer - Treatment history: Treatment-naive - Follow-up duration: Not explicitly mentioned, but primary endpoint was progression-free survival (PFS)

CDK4/6 Inhibitor

- Specific CDK4/6 inhibitor used: Palbociclib - Dosing and schedule: Palbociclib 125 mg daily (3 weeks of treatment, then 1 week off) - Combination therapy: Palbociclib plus letrozole (2.5 mg daily) - Treatment duration: Not explicitly mentioned - Comparator arm: Placebo plus letrozole (2.5 mg daily)

Signaling Changes

- Cell cycle pathway changes: CDK4/6 inhibition prevents RB phosphorylation, arresting cell cycle progression. - Growth factor signaling alterations: Increased FGFR2 and ERBB3 expression associated with greater PFS gain. - Transcriptional changes: Higher ESR1 expression associated with greater benefit from palbociclib and letrozole. - Protein expression changes: Higher CDK4 levels associated with endocrine resistance. - Immune signaling changes: PD-1 signaling pathway associated with reduced PFS benefit.

Biomarker Findings

- Predictive biomarkers: Higher CDK4 levels associated with endocrine resistance; lower PD-1 levels associated with greater benefit from palbociclib plus letrozole. - Prognostic biomarkers: Higher ESR1 gene expression associated with greater benefit from both placebo plus letrozole and palbociclib plus letrozole. - Pharmacodynamic biomarkers: Association of CDK4 levels with endocrine resistance suggests a role in drug activity. - Biomarker type: Gene expression (CDK4, PD-1, ESR1, FGFR2, ERBB3); protein levels (RB). - Sample source: Tumor tissue (FFPE samples). - Quantitative results: Higher CDK4 levels associated with shorter PFS in placebo arm; statistical significance in associations with PFS. - Clinical correlation: Biomarkers associated with PFS gains and response to treatment.

Resistance Mechanisms

- Intrinsic resistance mechanisms: Higher CDK4 levels associated with endocrine resistance; lower PD-1 levels associated with greater benefit. - Molecular alterations associated with resistance: Hypothesized RB loss and CCNE1 amplification, though incidence is low. - Proposed strategies to overcome resistance: Targeting interplay between steroid hormone and peptide growth factor signaling. - Temporal aspects: Not explicitly discussed.

Clinical Correlations

- Response rates by molecular subgroup: Palbociclib plus letrozole confers efficacy on both luminal A and B patients. - Progression-free survival associations with biomarkers/pathway changes: Higher CDK4 levels associated with endocrine resistance; lower PD-1 levels associated with greater benefit; active growth factor signaling (FGFR2, ERBB3) associated with greater PFS gain. - Overall survival correlations: Not mentioned. - Toxicity correlations with molecular alterations: Not mentioned. - Predictive accuracy of molecular markers: Not explicitly detailed. - Clinical utility assessments (sensitivity, specificity, positive/negative predictive values): Not explicitly detailed.

Analysis Methods

- Sample collection timing: Baseline - Sample types analyzed: FFPE tumor samples - Analytical techniques used: RNA-seq (EdgeSeq Oncology Biomarker Panel), Immunohistochemistry (IHC), Fluorescence in situ hybridization (FISH) - Validation methods: Cox regression analysis, log-rank test with multiple testing corrections (Benjamini-Hochberg procedure) - Statistical approaches for biomarker analysis: Hypothesis-driven and data-driven approaches, Cox regression analysis - Quality control measures: >70% tumor content, <20% necrosis

Purpose: Preclinical data identified the cyclin-dependent kinase 4/6 (CDK4/6) inhibitor palbociclib as synergistic with antiestrogens in inhibiting growth of hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2−) human breast cancer models. This observation was validated clinically in the randomized, placebo-controlled, phase III PALOMA-2 study. Experimental Design: To determine markers of sensitivity and resistance to palbociclib plus letrozole, we performed comprehensive biomarker analyses, investigating the correlation with progression-free survival (PFS), on baseline tumor tissues from PALOMA-2. Results: Despite a broad biomarker search, palbociclib plus letrozole demonstrated consistent PFS gains versus placebo plus letrozole, with no single biomarker or cassette of markers associated with lack of benefit from combination treatment. Palbociclib plus letrozole confers efficacy on both luminal A and B patients. Higher CDK4 levels were associated with endocrine resistance which was mitigated by the addition of palbociclib, whereas lower PD-1 levels were associated with greater palbociclib plus letrozole benefit. Tumors with more active growth factor signaling, as exemplified by increased expression of FGFR2 and ERBB3 mRNA, appeared to be associated with greater PFS gain from the addition of palbociclib. Conclusions: These data underscore the importance of CDK4/6 signaling in HR+/HER2− breast cancer and suggest that the interplay between steroid hormone and peptide growth factor signaling could drive dependence on CDK4/6 signaling. See related commentary by Anurag et al., p. 3

Introduction

The molecular heterogeneity of breast cancer is well established (1), but breast cancer is still approached clinically as three large therapeutic subgroups: hormone receptor-positive (HRþ), human epidermal growth factor receptor 2-amplified (HER2þ), and the so-called triple-negative breast cancer (TNBC), defined by the lack of hormone receptors and HER2 amplification/overexpression. Approximately 65% of breast cancers fall into the HRþ/HER2À category (2,3). The standard treatment for these patients has been hormonal blockadebased therapies directed at inhibiting the estrogen/estrogen receptor (ER) signaling pathway by various modalities (4). However, it is well understood that a subset of women with HRþ/HER2À disease will present with either de novo or acquired resistance to endocrine-based therapies (5). Numerous signaling pathways have been implicated as possible mechanisms for resistance, including aberrant peptide growth factor signaling pathways mediated by the epidermal growth factor, insulin growth factor, fibroblast growth factor, and other pathways (6). In addition, alterations in intracellular signaling pathways such as the PI3K, MAPK, Src, and others have been implicated in endocrine resistance in laboratory studies, and many of these alterations are correlated with poor clinical outcomes (7).

Therapeutic targeting of most of these signaling pathways in the frontline setting of advanced HRþ/HER2À breast cancer has not yielded significant improvements in outcomes. In 2015, the approval of palbociclib, a small-molecule inhibitor of CCND1/CDK4 kinase activity and CDK6 (a homolog of CCND1/CDK4 kinase activity; ref. 8), in combination with letrozole represented a breakthrough in the management of women with HRþ/HER2À advanced breast cancer (ABC). This was the first molecularly targeted agent that demonstrated a significant improvement in outcomes for this group of women (9,10). These data were initially met with some skepticism given the lack of specific biomarkers, other than ER-positivity (ERþ), as a way to identify patients who would receive benefit from treatment. However, since then, multiple phase III studies have validated the clinical benefit of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors in HRþ/HER2À breast cancers (11)(12)(13).

Cyclin-dependent kinases 4 and 6, together with the protein cyclin D1 (CCND1), are critical regulators of cell-cycle progression (14). Preclinical studies have shown that CDK4/6 inhibition prevents DNA replication by arresting progression from the G 1 to the S phase during cell division, thereby preventing tumor cell proliferation (8,15). The CDK4/6-CCND1 complex phosphorylates the retinoblastoma (RB) protein, a tumor suppressor, releasing the E2F and DP transcription factors that regulate expression of genes required for entry into S phase of the cell cycle (16,17). CDK4/6 activity and progression to the G 1 phase are negatively regulated by the CDK4/6-interacting proteinkinase inhibitory protein (Cip-Kip) and by the inhibitor of the cyclindependent kinase 4 (INK4) families, typified by p16, the protein product of CDKN2A (16,18).

Using a panel of 47 human breast cancer and immortalized cell lines, we found that ERþ models were most sensitive to growth inhibition by palbociclib (15). This included data from a cell line that was antiestrogen-resistant (15). The PALOMA-1 study was an openlabel, randomized phase II trial designed to validate this observation (9). The study prospectively enrolled women with advanced frontline ERþ/HER2À breast cancer and included two distinct cohorts of patients; cohort 1 enrolled patients based solely on ERþ/HER2À status, whereas cohort 2 further restricted eligibility to enroll ERþ/HER2À patients whose tumor also contained CCND1 amplification and/or CDKN2A (p16) loss (9). Findings from this study were consistent with the preclinical studies and were the first to clinically demonstrate a role for CDK4/6 inhibitors in ERþ breast cancer. The biomarker analysis of the PALOMA-1 study failed to demonstrate a relationship between CCND1 amplification or p16 loss and efficacy of palbociclib in combination with letrozole (9). Consequently, the pivotal phase III PALOMA-2 study enrolled ABC patients who had not received prior treatment for advanced disease using only ERþ/HER2À status, without further biomarker enrichment (11). A total of 666 postmenopausal patients were enrolled to receive palbociclib plus letrozole or placebo plus letrozole (11); the addition of palbociclib to letrozole resulted in a statistically significant, robust, and clinically meaningful 13-month improvement in median progression-free survival (PFS) compared with placebo plus letrozole [27.6 months vs. 14.5 months; HR, 0.56; 95% confidence interval (CI), 0.46-0.69; P < 0.0001; data cutoff date: May 31, 2017; ref. 19] . This benefit was consistent across patients with various clinical parameters, including disease-free interval >5 years, nonmeasurable or measurable disease, visceral or nonvisceral disease, and bone-only disease, as well as those who had received prior endocrine therapy (19).

In an attempt to garner further information on a population of patients who received benefit from the addition of a CDK4/6 inhibitor in ERþ/HER2À breast cancer, baseline tumor tissues were required for enrollment in the PALOMA-2 study. Using these tissues, we performed a comprehensive preplanned assessment evaluating additional DNA, mRNA, and protein biomarkers and correlated them with the clinical outcomes observed in the PALOMA-2 study.

PALOMA-2 study design

The details of the PALOMA-2 study have been previously published (11). Briefly, it was a double-blind, phase III placebocontrolled study that randomized patients 2:1 to receive palbociclib 125 mg daily (3 weeks of treatment, then 1 week off) plus letrozole (2.5 mg daily) or placebo plus letrozole (2.5 mg daily) in postmenopausal women who had not received prior systemic therapy for ERþ/HER2À ABC. This study was approved by an Institutional Review Board or equivalent ethics committee at each study site. The primary endpoint was investigator-assessed PFS. The study was conducted in accordance with the International Conference on Harmonization Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki, and all patients provided written informed consent before enrollment.

Tissue samples

The submission of formalin-fixed paraffin-embedded (FFPE) tumor samples was mandatory in PALOMA-2. Patients consented to the assessment of biomarkers associated with sensitivity and/ or resistance to palbociclib plus letrozole per study protocol. If an FFPE tissue block could not be provided, a minimum of 12 glass slides, each containing an unstained 5-mm FFPE tissue section, were required for analysis. Whenever possible, recently biopsied tissue samples from a metastatic or recurrent tumor lesion were provided. If archival tissue was unavailable, a new biopsy was required.

Gene expression analysis

Analyses of gene expression (RNA) were performed by using the EdgeSeq Oncology Biomarker Panel (HTG Molecular Diagnostics, Inc). This system uses targeted capture sequencing to quantitate RNA expression levels of gene targets in FFPE tissues. The first section of breast cancer FFPE tissue was stained with hematoxylin and eosin (H&E). A board-certified pathologist assessed the tumor cell content and tissue necrosis. Tumor content was estimated based on the number of malignant cells as a percentage of all cells (i.e., malignant plus normal cells in the tissue section). The acceptance criterion for analysis was set at >70% of tumor content. Necrosis was assessed based on the percentage of necrotic tissue within the total tissue area. The acceptance criterion for analysis was set at <20% necrosis. Macrodissection was performed on the tissue sections if the tumor content was <70% or if necrosis was !20%. Sample preparation was conducted following laboratory processes and manufactory protocols. Sequencing was performed on an Illumina NextSeq 500 Sequencer (Illumina, Inc.).

Translational Relevance

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have demonstrated significant activity in estrogen receptorpositive/human epidermal growth factor receptor 2-negative (ERþ/HER2-) advanced breast cancer. Further biomarkers to refine the population beyond ERþ/HER2-that benefits remain to be identified. We performed a comprehensive biomarker analysis using patient tissues from PALOMA-2, a randomized phase III study of palbociclib-letrozole versus placebo-letrozole. These data were correlated with progression-free survival (PFS) to determine markers of sensitivity and resistance to the combination. Palbociclib-letrozole demonstrated consistent PFS gains versus placeboletrozole. No single biomarker or cassette of markers was associated with lack of benefit from combination treatment. Our findings support the mechanism that the interplay between two growthpromoting pathways (steroid hormone and peptide hormone signaling pathways) drives CDK4/6 dependence, resulting in clinical benefit from palbociclib-letrozole. These results may guide additional therapeutic options in patients with ERþ/HER2advanced breast cancer and contribute to our understanding of appropriate patient selection.

To normalize the sequencing data, probe counts were transformed into log 2 counts per million. Expression values were quantile normalized. HTG Molecular Diagnostics, Inc., was blinded to patient information and clinical outcomes.

Molecular subtype classification

Given the lack of large diverse reference tumor sets profiled with the EdgeSeq Oncology platform and the fact that only ERþ patients were included in the PALOMA-2 study, we could not use the PAM50 classification scheme, which determines subtype relative to a baseline of heterogeneous tumors from the same cohort or from a comparable reference database (20). Therefore, the single sample predictor algorithm Absolute Intrinsic Molecular Subtyping (AIMS) was adopted to classify subtypes through a set of binary rules that compare expression measurements for pairs of genes from each patient independently (21). As only 42 of the 100 binary rules could be applied based on genes in the EdgeSeq Oncology BM panel, classification performance was assessed by downsampling TCGA data from genome-wide to the EdgeSeq Oncology panel subset. Using all genes versus EdgeSeq Oncology panel genes only, the agreement with PAM50 classification was 77% versus 76%, respectively.

Exploratory unbiased discovery statistical analysis

A data-driven exploratory unbiased discovery analysis was performed for gene expression biomarkers predictive of the benefit from palbociclib plus letrozole. Using Cox regression analysis, the search first identified genes whose expression was significantly associated with PFS as a continuous variable within the palbociclib plus letrozole or placebo plus letrozole arms (FDR < 0.1), followed by a cross-arm interaction analysis both before and after accounting for known clinical-pathological factors (i.e., site of disease, prior hormone therapy, disease-free interval, age, race, region, baseline Eastern Cooperative Oncology Group status; Supplementary Table S1 ). To further evaluate underlying biological processes mediating palbociclib plus letrozole response, we transformed data from gene to pathway activity using MSigDB curated (c2) gene sets and a Gene Set Variation Analysis (GSVA) algorithm (22). Cox regression analysis as described for the genelevel search indicated above and then carried out with pathway activity scores.

Protein expression analysis by IHC

Expressions of ER (Dako 1D5 anti-ER antibody), RB (BD Biosciences G3-245 anti-RB antibody), CCND1 (Biocare Medical SP4 anti-Cyclin D1 antibody), p16 (Ventana E6H4 anti-p16 antibody), and Ki67 (Ventoan 30-9 anti-Ki67 antibody) were analyzed retrospectively at a Clinical Laboratory Improvement Amendmentscertified laboratory using validated IHC assays. The results were quantified using H-score methodology (3 Â percentage of strongly staining nuclei þ 2 Â percentage of moderately staining nuclei þ percentage of weakly staining nuclei, giving a range of 0-300). For ER, CCND1, and p16, positive expression was defined as an H-score of !1.

RB was reported as positive if !5% of tumor cells exhibited a positive staining reaction, in a nuclear pattern (Vision Biosystems). The staining intensity of tumor cells was reported based on the following guidelines: 0 ¼ nuclear staining not detectable, 1þ ¼ nuclear staining translucent, 2þ ¼ nuclear staining opaque, and 3þ ¼ nuclear staining solid.

FISH analysis

FISH was performed using a 4-color FISH test to detect both CCND1 amplification and CDKN2A (p16) deletion simultaneously from a single tissue section. A minimum of two 5-mm FFPE tissue sections were used for this assay. The first section was stained with H&E to visualize the tumor compartment within the tissue. The second and subsequent sections were reserved for FISH analysis. A 4-color FISH probe set was hybridized to the section. Using the H&E as a guide, the invasive component of the tumor was analyzed for CCND1 amplification and CDKN2A deletion. A minimum of 20 cells were scored in each case.

The DNA probes [CCND1 (Spectrum Red), CEP11 (Spectrum Green), CDKN2A (Spectrum Gold), and CEP9 (Spectrum Qua)] were purchased from Abbott Molecular. Manual visualization and interpretation of fluorescence signals were performed using an Olympus BX51 Fluorescence microscope. CCND1 amplification was quantified by CCND1/CEP11 ratio, with a cutoff of >1.5 considered positive. The CDKN2A deletion was quantified by CDKN2A/CEP9 ratio, with a cutoff of <0.8 considered positive. Neogenomics Laboratories, Inc., was blinded to patient information and clinical outcomes.

Statistical analysis

Baseline characteristics and demographics in the biomarker analysis set were summarized by treatment arms. Analyses of investigator-assessed PFS were performed in the subgroups, defined by corresponding baseline biomarker tests using the Kaplan-Meier method and Cox proportional hazard models to investigate potential association between biomarker levels and PFS from palbociclib plus letrozole or placebo plus letrozole treatments. Biomarker analyses were performed using both hypothesis-driven (supervised) and data-driven (unsupervised) approaches. Results were visualized using Kaplan-Meier plots by median or quantile dichotomization. A log-rank test was used to compare PFS differences, with multiple testing corrections made and FDRs based on the Benjamini-Hochberg procedure.

Results

Consistent PFS benefit in the biomarker and intent-to-treat populations A total of 568 of 666 (85%) baseline tumor tissues from the 666 patients randomized were collected: 379 of 444 patients (85%) in the palbociclib plus letrozole arm and 189 of 222 patients (85%) in the placebo plus letrozole arm. A total of 455 of 568 (80%) patient tissues were evaluated using gene expression analyses, 520 (92%) were evaluated by FISH, and 568 (100%) were evaluated using IHC (Supplementary Fig. S1 ). Baseline patient demographics and clinical characteristics were similar between the gene expression, IHC, and FISH biomarker subsets and the overall PALOMA-2 intent-to-treat (ITT) population (Table 1 ). Of note, the PFS benefit associated with palbociclib plus letrozole was similar between the various biomarker subsets defined below, as well as in the overall ITT population (Supplementary Fig. S2 ).

CDK6, cyclin D, cyclin E, RB, and p16 expression levels do not predict the benefit of palbociclib in combination with letrozole by IHC, FISH, or gene expression analysis

Analyzing genes involved in the cyclin D-CDK4/6-Rb pathway showed that irrespective of the baseline gene expression levels (higher or lower based on dichotomization by median) of CCND1, CCNE1/2, CDK2/6, RB1, CDKN2A, or ESR1, the addition of palbociclib to letrozole demonstrated a benefit versus placebo plus letrozole, in line with the overall ITT population (Fig. 1A ). Similar results were obtained when gene expression levels were stratified by upper and lower quartiles (Supplementary Fig. S3 ). Similar results were observed for the protein levels of ER, RB, CCND1, and p16 when expression was assessed by quartiles using an H-score analysis (Fig. 1B ). As seen in PALOMA-1 and based on copy-number (FISH) analysis from the current study, patients benefited from the addition of palbociclib to letrozole regardless of whether their tumor contained CCND1 amplification and/or CDKN2A (p16) loss (Fig. 1C ).

Concordance analysis showed a reasonable agreement between protein and mRNA expression for ER, CCND1, and Ki67 (Spearman R ¼ 0.50, 0.39, and 0.48, respectively), but weaker correlation for RB (Spearman R ¼ 0.22) and p16 (R ¼ 0.14; Supplementary Table S2 ). In addition, the protein levels of ER, CCND1, and RB were most correlated with their own transcript among all genes across the EdgeSeq Oncology panel, whereas the cis-correlation for Ki67 was ranked fifth panel wide, demonstrating the high specificity of the relationship. It is also worth noting that the concordance between the expression by IHC and amplification by FISH analyses of CCND1 (coefficient R ¼ 0.19) and p16 (R ¼ 0.09) was also very low (Supplementary Table S3 ).

The incidence of RB loss in ERþ breast cancer is low (<5%; ref. 23). In our cohort, total RB expression as assessed by IHC was positive in 90.9% (512/563) of patients and negative in 9.1% (51/ 563) of patients. In patients with RB-positive (RBþ) tumors, the observed HR from the unstratified analysis was 0.543 (95% CI, 0.433-0.681; log-rank P < 0.0001 in favor of palbociclib plus letrozole). The RB-"negative" (RBÀ) subset had a weaker HR of 0.868 (95% CI, 0.424-1.777, log-rank P ¼ 0.698), but this is clearly limited by the small numbers of patients in this cohort. Similarly, we analyzed RB gene expression based on a cutoff of 8 from negative control probes (Fig. 2A ) as well as dichotomized by the median (Fig. 2B ) and by quartile (Fig. 2C ) to define RB higher versus lower expression between the two treatment groups. Exploratory assessments of PFS by RB1 gene using this approach revealed a consistent benefit with palbociclib plus letrozole. Of note, there was no evidence of palbociclib benefit in truly RB-null tumors (Fig. 2 ; Supplementary Fig. S4 ).

CDK4 expression levels predict resistance to placebo plus letrozole but not to palbociclib plus letrozole

Contrary to results with the other cell-cycle genes and proteins analyzed above, higher CDK4 gene expression level was associated with an important clinical parameter measured in the study. These patients demonstrated shorter PFS with placebo plus letrozole (P ¼ 0.000972 and P ¼ 0.000779 when gene expression levels were treated as a continuous variable or dichotomized by median, respectively; Fig. 3A ; Supplementary Fig. S5 ). This relationship was not significant in the palbociclib plus letrozole arm (P ¼ 0.91 and P ¼ 0.13 by continuous and median analysis, respectively). As noted above, the expression of CDK6 did not have a similar effect (lower expression, HR ¼ 0.596; higher expression, HR ¼ 0.592; Fig. 1A ).

ESR1 expression levels are associated with longer PFS in both placebo plus letrozole and palbociclib plus letrozole arms

Higher ESR1 gene expression was found to be associated with greater benefit in both the placebo plus letrozole and the palbociclib plus letrozole arms (Fig. 3B ), underscoring the importance of ER in predicting response to both letrozole and its combination with palbociclib. Importantly, in both the ESR1 high-and low-expression groups, the addition of palbociclib increased PFS by approximately 10 months (11.2 to 21.5 months for the lower ESR1 expression level and 18.2 to 27.7 months for the higher ESR1 expression level). This is consistent with protein data showing that regardless of the expression level of ER

Finn et al.

Clin Cancer Res; 26(1) January 1, 2020 CLINICAL CANCER RESEARCH 114 protein in the tumor as measured by IHC, patients with ERþ disease benefited from palbociclib plus letrozole over placebo plus letrozole (Fig. 1B ).

Both luminal A and B subtypes determined by either Ki67 or gene expression-based profiles benefit from the addition of palbociclib to letrozole

We explored whether the benefit of palbociclib plus letrozole may differ by intrinsic breast cancer subtype. Based on published literature, Ki67 protein cutoffs of >20% (24,25) or >15% (26,27) were used to subdivide tumors into luminal B versus A subtypes. In total, 58% or 46% of patients with Ki67 IHC data available had luminal A (Ki67 20% or 15%), and 42% or 54% of patients had luminal B tumors (Ki67 >20% or >15%), respectively. Analyses using either cutoff led to the observation that patients with either luminal A or B subtype benefited from treatment with palbociclib plus letrozole compared with placebo plus letrozole (Supplementary Fig. S6 ). Tumors were also classified according to their mRNA profiles using the AIMS algorithm (21). This analysis demonstrated that 50% of study patients had luminal A (n ¼ 229), and 30% had luminal B tumors (n ¼ 135; Fig. 4A ). Gene expression-based analysis also showed that patients of either luminal subtype benefited from the addition of palbociclib to letrozole (luminal A: HR, 0.55; 95% CI, 0.39-0.77; P ¼ 0.000547 and luminal B: HR, 0.51; 95% CI, 0.34-0.77; P ¼ 0.00109; Fig. 4B and C ). The number of patients with nonluminal molecular subtypes were smaller, with the largest group being the HER2Àlike subtype (19%, n ¼ 85), and the study only had very small numbers of patients with basal-like (n ¼ 2) and normal-like (n ¼ 4) subtypes (Fig. 4A ); these groups were not large enough for a formal analysis. The increase in median PFS with the addition of palbociclib in the HER2Àlike group was less than in the luminal subgroups, but again, these observations are limited by the small group size.

PALOMA-2 enrolled patients with ERþ/HER2À tumors based on the local site assessments. Although centralized FISH analysis was not performed, we confirmed the lack of HER2 amplification by examining the mRNA expression of the ERBB2 gene itself and found that it did not show a bimodal distribution, with exceptionally high outliers typically associated with amplified tumors (28).

High level of fibroblast growth factor receptor 2 and Erb-B2 receptor tyrosine kinase 3 predicts greater degree of benefit from palbociclib plus letrozole

In addition, we performed a panel-wide unsupervised analysis to identify potential predictive biomarkers of benefit in each arm of the study. After correcting for multiple hypothesis testing, 9 and 16 candidate genes were identified from the palbociclib plus letrozole and placebo plus letrozole arms, respectively, using an FDR of <0.1 for within-arm PFS association and a cross-arm gene expression treatment interaction of P < 0.1 (Supplementary Table S1 ). This was performed both before and after accounting for known clinical-pathological factors, resulting in a total of 12 relative resistance markers (CHI3L1, PDCD1, PHGDH, PORCN, LPL, PIK3R5, STEAP4, NUMB, ATF6, TNFRSF25, HHAT, and BMPR1B) and 13 relative sensitivity markers (SMAD2, TIRAP, SETBP1, KRT19, GSTM3, SORD, CYR61, CDK4, EZH2, MDK, THBS2, NNMT, and TMSB10). Importantly, as noted in the supervised approach, CDK4 expression was significantly associated with an enhanced benefit from palbociclib plus letrozole. Interestingly, tumors with higher levels of pretreatment programmed cell death protein 1 (PDCD1) mRNA expression tended to receive less benefit from addition of palbociclib to letrozole than those with lower levels (FDR ¼ 0.099, interaction P ¼ 0.020 from continuous analysis; Fig. 5A ).

In addition to individual genes, we also assessed whether any biologically defined gene signatures might predict benefit with palbociclib plus letrozole by summarizing the data in thousands of MSigDB gene sets using the GSVA algorithm (22). Analogous Cox regression analysis highlighted the PD-1 signaling pathway as being associated with reduced PFS benefit from the addition of palbociclib to letrozole (palbociclib plus letrozole, FDR ¼ 0.039 and interaction P ¼ 0.0893). Meanwhile, tumors with more active growth factor signaling appeared to be associated with greater PFS gain from the combination of palbociclib plus letrozole (FDR ¼ 0.027 and interaction P ¼ 0.0888), as exemplified by increased expression of FGFR2 and Erb-B2 receptor tyrosine kinase 3 (ERBB3; Fig. 5B and C ).

Discussion

The development of CDK4/6 inhibitors in breast cancer has followed a rational clinical development plan based on compelling preclinical observations indicating that having an ERþ luminal phenotype was the most predictive for response to this class of targeted therapy. The clinical benefit of the CDK4/6 inhibitor palbociclib in combination with endocrine therapy initially shown in the PALOMA-1 study (9) has now been confirmed in multiple randomized phase III clinical studies in HRþ/HER2À breast cancer using three different CDK4/6 inhibitors (11)(12)(13)(29)(30)(31). As a result, CDK4/ 6 inhibitors are now established as a standard-of-care option for both endocrine-sensitive and endocrine-resistant HRþ/HER2À metastatic breast cancer (32,33). Despite this observed clinical benefit, there remains interest in further defining the responsive population for efficacy with CDK4/6 inhibitors, given the side effects and costs associated with addition of a CDK4/6 inhibitor to endocrine therapy. Using tissues from a prospective phase III study (PALOMA-2), we performed a comprehensive molecular analysis of patient tissues at the DNA, mRNA, and protein levels in an attempt to identify biomarker(s) associated with benefit from the addition of palbociclib to letrozole for HRþ/HER2À breast cancer. Ultimately, our analysis underscores the importance of selecting patients that are ERþ; this subgroup appears to be dependent on signaling through the CDK4/6: cyclin D:RB pathway, given a dependence on estrogen signaling, and we have observed convergent actions on the CDK4/6:cyclin D:RB pathway in some patients with increased expression of receptor tyrosine kinases. In addition, proposed alterations that are likely associated with resistance to a CDK4/6 inhibitor (i.e., CCNE1 amplification or RB loss) appear to be rare events in this breast cancer subtype (23).

We initially focused our approach on potential biomarkers within the cyclin D:CDK4/6:RB pathway, including CDK4, CDK6, cyclin D, cyclin E, p16, and RB. Expression of these markers, as determined by IHC or mRNA levels, was not predictive of palbociclib benefit in combination with letrozole in this population, i.e., patients who had not received prior systemic therapy for ERþ/ HER2À ABC. When considering these data, it should be noted that an ERþ status already classifies these tumors into a higher RB expression level group (15), indicating that we are comparing relative RB levels within a "high RB expression" group. It has been hypothesized that amplification of CCNE1 or RB loss could be associated with resistance to CDK4/6 inhibitor effects (34). However, the incidence of these alterations in ERþ luminal breast cancer is rare and ( 23), therefore, the numbers of patients in PALOMA-2 with such alterations were not sufficient to validate this hypothesis. Similarly, when evaluating RB expression by IHC, the number of patients with truly RB "negative" status was small. Furthermore, the validation of this assay in tissues collected from multiple centers without controlled processing and fixation is limited in our analysis. There was minimal concordance between RB1 gene expression and RB protein level, indicating potential methodological limitations in the detection of RB in HRþ breast cancer. Based on these data, we could not conclude that palbociclib has activity in truly RB-null or CCNE1-amplified tumors.

Previous studies have reported CCND1 amplification in 29% of luminal A and 58% of luminal B tumors, and CDK4 gains in 14% CLINICAL CANCER RESEARCH and 25% of luminal A and luminal B tumors, respectively (35); low expression of p16 in luminal A tumors has also been reported (36).

The PALOMA-1 study independently assessed CCND1 amplification and/or p16 loss in an open-label randomized study (9). In the cohort of patients with CCND1 amplification or p16 loss (n ¼ 99), the benefit of palbociclib plus letrozole was similar to patients without those genomic changes (9). These observations are now confirmed in the current placebo-controlled PALOMA-2 study. The current results showed that CDK6, cyclin D, cyclin E, p16, and RB expression levels as measured here were not predictive of palbociclib benefit in combination with letrozole in this population of patients who had not received prior systemic therapy for ERþ/ HER2À ABC. Large randomized studies have consistently shown that CDK4/6 inhibitors add to endocrine therapy activity in HRþ/HER2-ABC, as evidenced by improvements in median PFS (9,(11)(12)(13). We also investigated endocrine resistance and sensitivity as potential markers of palbociclib activity. Our study demonstrated high concordance between quantitative IHC by H-score and ESR1 mRNA expression using data from contemporary care settings. These data suggest that high ESR1 expression may predict a potentially greater degree of benefit from letrozole in metastatic breast cancer and are consistent with previous literature supporting ER expression and its relationship to HER2 signaling (37). Preclinical data indicate that endocrine-resistant cell lines demonstrate high levels of CDK4 expression (38,39). Consistent with these observations, our findings showed that patients treated with placebo plus letrozole who have tumors expressing high levels of CDK4 expression exhibited shorter median PFS than patients with lower levels. These findings suggest that elevated levels of CDK4 may contribute to an endocrine resistance phenotype that can be circumvented with the addition of palbociclib. Thus, levels of ESR1 expression appeared to be prognostic, and CDK4 may be predictive of letrozole resistance (interaction P ¼ 0.016). These findings suggest that the effect of CDK4/6 inhibition may be independent of those exerted by the endocrine-mediated pathway.

Multiple growth steroid peptides signal and prompt tumor growth via the ER (40). Cyclin D is a direct downstream effector of ER signaling (17,41). From an unbiased panel-wide analysis, we demonstrated that more active growth factor signaling, including high levels of ERBB3 and FGFR2 expression, was associated with a larger PFS gain from the combination of palbociclib and letrozole. These data provide evidence that the interplay between steroid hormone and peptide growth factor signaling in ERþ breast cancer could drive dependence on CDK4/6, which leads to the greater clinical benefit seen with CDK4/6 inhibition through the addition of palbociclib to letrozole.

Genomic assays classify luminal disease into luminal A and luminal B subtypes. Published results in early breast cancer indicate that patients with luminal A status may derive more benefit from endocrine therapy than patients with luminal B disease (24,25,42). In the present study, Ki67 cutoffs (24,25,43) and the gene expression-based AIMS algorithm (21) were used to stratify luminal subtypes. Patients with both luminal A and B disease benefited to a similar degree from the addition of palbociclib to letrozole. There was less of a benefit associated with palbociclib plus letrozole in the HER2Àlike group; however, this was a relatively small subgroup, and further evaluation will be required to address this issue.

There is increasing interest in the development of immuneoncology agents in breast cancer, although to date, most efforts have focused on triple-negative disease (44). Preclinical studies have evaluated palbociclib combined with checkpoint inhibitors and shown potential efficacy suggesting effects of CDK4/6 inhibition in immune effector cells (15). Of note, an unsupervised analysis of the current study identified not only a higher level of PD-1 itself, but also that the pathway is associated with less benefit from the addition of palbociclib to letrozole versus lower expressors. PD-1 expression is a potential indication of lymphocyte infiltration, and emerging data suggest that low PD-1 expression levels may be prognostic of poor outcomes. This may represent a potential avenue to improve outcomes with intrinsic resistance to endocrine-CDK4/6 inhibitor-based therapy and warrant further clinical investigation. Ongoing studies are now evaluating immune checkpoint inhibitors targeting PD-1 in combination with CDK4/6 inhibitors, including palbociclib (NCT02778685).

One major limitation of the current analysis is that tissue samples could have been from either primary or metastatic biopsies. The origin of tissue samples was not recorded, and biomarkers may differ in samples from metastatic and primary biopsies. The impact of this was not assessed in the current study. In addition, host factors (e.g., pharmacogenomics) were not assessed in the present study, only tumor tissue factors. Despite these limitations, the current findings provide important biological insights into the interplay between the ER signaling, peptide growth factor signaling, and cell-cycle pathways that may ultimately help guide additional therapeutic opportunities in patients with ERþ/HER2-ABC. These findings may contribute to understanding appropriate patient selection, particularly in early breast cancer, where greater clinical benefit with this class of agents may occur. Large adjuvant studies of palbociclib in early stage breast cancer are ongoing (clinicaltrials. gov: NCT02513394, NCT03609047) and will provide additional tissues for biomarker correlates.

Acknowledgements

AcknowledgmentsThis study was sponsored by Pfizer Inc. Editorial support was provided by Anny Wu, PharmD, of Complete Healthcare Communications, LLC, a CHC Group company, and was funded by Pfizer Inc.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.Received March 4, 2019; revised July 1, 2019; accepted September 11, 2019; published first September 16, 2019.

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