Elicit: CDK4/6 Inhibition and G1 to S Arrest

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CDK4/6 Inhibition and G1 to S Arrest

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May 5, 2026

CDK4/6 inhibition cell-cycle arrest G1 to S palbociclib

Palbociclib consistently induces G1 to S cell cycle arrest through CDK4/6 inhibition and Rb pathway blockade, achieving complete arrest in 87-90% of Rb-proficient hormone receptor-positive breast cancers, though the arrest is cytostatic and requires continuous therapy to maintain.

Abstract

Palbociclib consistently induces G1 to S phase cell cycle arrest across multiple cancer types through inhibition of CDK4/6, preventing retinoblastoma (Rb) phosphorylation and suppressing E2F-target gene expression. In neoadjuvant trials of hormone receptor-positive breast cancer, complete cell cycle arrest (Ki-67 ≤2.7%) was achieved in 87-90% of patients when palbociclib was combined with endocrine therapy, representing a threefold increase over endocrine therapy alone. This profound antiproliferative effect translated to clinically meaningful progression-free survival benefits in advanced disease, with median PFS of 9.2 months versus 3.8 months for combination therapy versus endocrine therapy alone (HR 0.42). However, the arrest is cytostatic rather than cytotoxic—concurrent suppression of apoptosis limits tumor regression, and continuous therapy is necessary to maintain cell cycle arrest as discontinuation allows Ki-67 rebound. Mechanistically, response depends on intact Rb function, with RB1 loss conferring complete resistance, while high cyclin E levels and elevated CDK2 activity enable G1/S bypass. Sustained mTORC1 activity during palbociclib treatment converts reversible arrest to irreversible senescence, and autophagy induction serves as an adaptive resistance mechanism that can be targeted through combination strategies.

The clinical benefit of palbociclib-induced G1 arrest is biomarker-dependent and context-specific. Tumors with intact Rb, absent or low cyclin E (particularly low-molecular-weight isoforms), and adequate p27 levels demonstrate maximal sensitivity, with progression-free survival exceeding 400 months in optimally selected populations. Grade 3/4 neutropenia occurs in 51-62% of patients but is largely asymptomatic, with febrile neutropenia rates of only 0.6% and treatment discontinuation rates of 2.6%. Combination strategies targeting autophagy, PI3K/MEK signaling, or mTORC1 can overcome resistance mechanisms in biomarker-selected populations. The evidence demonstrates that palbociclib reliably induces G1 arrest in Rb-proficient cells, but durable clinical benefit requires continuous dosing, appropriate biomarker selection, and in some contexts, rational combination therapy to prevent adaptive resistance.

Methods

We analyzed 10 sources from an initial pool of 200, using 8 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Palbociclib Focus, Cell Cycle Measurement, Study Model Type, Control Groups, Study Type, Measurable Outcomes, Publication Type, Sample Size

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: “CDK4/6 inhibition cell-cycle arrest G1 to S palbociclib”

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 setting for CDK4/6 inhibition research, including:

Extract complete details about CDK4/6 inhibitor treatment regimen, including:

Extract all measurements of G1 to S cell cycle arrest and related markers, including:

Extract mechanistic findings related to CDK4/6 inhibition and G1/S arrest, including:

Extract clinical efficacy outcomes specifically related to CDK4/6 inhibitor treatment, including:

Extract findings about cellular consequences of CDK4/6-induced G1 arrest, including:

Extract factors affecting response to CDK4/6 inhibition and G1/S arrest, including:

Extract safety and tolerability data for CDK4/6 inhibitor treatment, including:

Results

Characteristics of Included Studies

The systematic review included 10 studies examining CDK4/6 inhibition by palbociclib and its effects on G1 to S phase cell cycle arrest. Studies comprised 6 clinical trials and 4 preclinical investigations. Full text was available for 6 studies, while 4 were assessed from abstracts only.

Study

Full Text Retrieved?

Study Type

Cancer Type and Subtype

Sample Size

Setting

Ma et al., 2017

Yes

Phase II neoadjuvant clinical trial

ER+/HER2- breast cancer

50 patients (18 premenopausal, 32 postmenopausal)

Clinical stage II-III patients

Johnston et al., 2019

Yes

Phase II randomized trial

ER+/HER2- breast cancer

307 patients

Postmenopausal women with primary tumors ≥2.0 cm

Arnedos et al., 2018

No

Randomized clinical trial

Early breast cancer (93% HR+, 8% HER2+)

74 palbociclib, 26 control

Early breast cancer patients

Maskey et al., 2020

Yes

Preclinical, in vitro

ER+ breast cancer

Multiple cell lines (MCF7, T47D, CAMA1)

ER+ breast cancer cell lines

Turner et al., 2015

Yes

Phase 3 clinical trial

Advanced HR+/HER2- breast cancer

521 patients

Pre- and postmenopausal women with relapsed/progressed disease

Vijayaraghavan et al., 2017

Yes

Preclinical (in vitro, in vivo) and clinical cohort

ER+ breast cancer and other solid tumors

109 patients in clinical cohort; multiple cell lines and xenografts

Cell lines (MCF7, T47D, ZR75-1), xenografts, advanced ER+ breast cancer patients

DeMichele et al., 2014

No

Phase II clinical trial

Advanced breast cancer (84% HR+/HER2-, 5% HR+/HER2+, 11% HR-/HER2-)

37 patients

Metastatic breast cancer, Rb+ with measurable disease

Tien & Sadar, 2021

No

Preclinical

Castration-resistant prostate cancer

Not mentioned

Human xenografts and cultured cells

Asghar et al., 2017

Yes

Preclinical

Triple-negative breast cancer (TNBC), LAR subtype

Multiple cell lines and xenografts

TNBC cell lines and MDA-MB-453 LAR xenografts

Kumarasamy et al., 2020

No

Preclinical

ER+ breast cancer and pancreatic cancer

Not mentioned

ER+ xenografts and pancreatic cancer PDX models

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Clinical trials predominantly focused on hormone receptor-positive breast cancer, with sample sizes ranging from 37 to 521 patients. The two largest trials were phase 3 (Turner et al., 521 patients) and phase 2 (Johnston et al., 307 patients). Neoadjuvant trials examined palbociclib in combination with endocrine therapy, while the advanced disease trial combined palbociclib with fulvestrant. Preclinical studies utilized established cell lines and xenograft models to investigate mechanistic aspects of CDK4/6 inhibition.

Treatment Regimens

Palbociclib dosing across studies followed established protocols, with clinical trials consistently using 125 mg daily on a 21-days-on, 7-days-off schedule, while preclinical studies utilized concentrations ranging from 500 nM to 1 μM in vitro.

Study

Palbociclib Dose and Schedule

Duration

Combination Therapy

Route

Ma et al., 2017

125 mg daily, days 1-21 of 28-day cycle

Four 28-day cycles, plus optional 10-12 day cycle 5

Anastrozole 1 mg daily; goserelin if premenopausal

Oral

Johnston et al., 2019

125 mg/day, 21-days-on, 7-days-off

14 weeks

Letrozole

Oral

Arnedos et al., 2018

125 mg daily

14 days until day before surgery

None mentioned

Oral

Maskey et al., 2020

500 nM

48 hours and various durations

Approved with endocrine therapies clinically, not specified in study

Added to cell culture medium

Turner et al., 2015

Not mentioned

Not mentioned

Fulvestrant; goserelin for pre/perimenopausal women

Not mentioned

Vijayaraghavan et al., 2017

In vitro: ≤1 μM for 6 days; In vivo: 25 mg/kg/day for 7 days

In vitro: 6 days treatment + 4 days recovery; In vivo: 7 days

Autophagy inhibitor HCQ

Oral gavage (in vivo)

DeMichele et al., 2014

125 mg orally, days 1-21 of 28-day cycle

Not mentioned

Not mentioned

Oral

Tien & Sadar, 2021

Not mentioned

Not mentioned

EPI-7170 (sequential or concomitant)

Not mentioned

Asghar et al., 2017

In vitro: 500 nM; In vivo: 50 mg/kg daily

In vitro: ≥2 weeks; In vivo: 21 consecutive days

Pictilisib, taselisib, AZD2014

Oral (in vivo)

Kumarasamy et al., 2020

Not mentioned

Not mentioned

MEK inhibitor in pancreatic cancer PDX models

Not mentioned

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The neoadjuvant studies demonstrated the importance of treatment duration and continuity. Ma et al. showed that Ki67 levels rebounded at surgery following palbociclib washout, but this rebound was suppressed by an additional cycle 5 of palbociclib immediately before surgery, suggesting continuous therapy may be necessary to maintain antiproliferative effects.

Cell Cycle Arrest and Proliferation Markers

Palbociclib consistently induced profound suppression of cellular proliferation across studies, with Ki-67 serving as the primary biomarker for cell cycle arrest.

Study

Ki-67 Baseline

Ki-67 Post-Treatment

% Change/CCCA Rate

Time Points

Other Markers

Ma et al., 2017

C0D1

C1D1 (anastrozole alone), C1D15 (palbociclib added)

CCCA rate: 26% (C1D1) vs 87% (C1D15)

Baseline, C1D1, C1D15, surgery

PAM50 11-gene proliferation score

Johnston et al., 2019

Baseline

14 weeks

Median log-fold change: -4.1 (palbociclib+letrozole) vs -2.2 (letrozole); Geometric mean: -97.4% vs -88.5%; CCCA: 90% vs 59%

Baseline, 2 weeks, 14 weeks

None mentioned

Arnedos et al., 2018

Day 1

Day 15

Antiproliferative response: 58% (palbociclib) vs 12% (control)

Day 15

Phospho-Rb

Maskey et al., 2020

Not mentioned

48 hours

Not mentioned

48 hours for DNA content

Cell cycle phase distribution measured using DAPI staining

Turner et al., 2015

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Vijayaraghavan et al., 2017

Not mentioned

After treatment and recovery phases

Palbociclib induced G1 arrest; CCCA achieved in 90% at appropriate doses

Not mentioned

BrdU (decreased in palbociclib-treated cells); Cell cycle phase distribution by propidium iodide

DeMichele et al., 2014

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Tien & Sadar, 2021

Not mentioned

Not mentioned

Doubling time increased to >63 hours vs 25 hours (control)

Not mentioned

Cell cycle phase distribution: combination prevented G1 and G2-M progression, caused S-phase arrest

Asghar et al., 2017

Not mentioned

48 hours post-treatment

Cell cycle length: 20 hours (vehicle) vs 38 hours (palbociclib) in CDK2 high cells

2 hours post-cytokinesis, 48 hours post-treatment

CDK2 activity reporter

Kumarasamy et al., 2020

Not mentioned

Not mentioned

Not mentioned

Not mentioned

Not mentioned

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The neoadjuvant trials provided the most detailed quantitative assessment of proliferative suppression. In the NeoPalAna trial, the complete cell cycle arrest rate (defined as Ki67 ≤2.7%) increased dramatically from 26% with anastrozole monotherapy to 87% after adding palbociclib. Similarly, Johnston et al. demonstrated that 90% of patients achieved complete cell cycle arrest with palbociclib plus letrozole compared to only 59% with letrozole alone. The median log-fold change in Ki-67 was significantly greater with combination therapy (-4.1 vs -2.2), corresponding to near-complete suppression of proliferation (-97.4% vs -88.5% geometric mean change). These findings indicate that CDK4/6 inhibition substantially enhances the antiproliferative effects of endocrine therapy beyond what can be achieved with endocrine therapy alone.

Mechanistic Foundations of Cell Cycle Arrest

Multiple studies investigated the molecular mechanisms by which palbociclib induces G1 arrest, with particular focus on the Rb pathway and downstream effectors.

Rb Pathway Modulation

Retinoblastoma (Rb) phosphorylation emerged as a central mechanistic determinant. Arnedos et al. demonstrated that palbociclib treatment led to significantly greater decreases in phospho-Rb compared to control, and changes in Ki67 correlated with changes in phospho-Rb (Spearman r=0.41). This relationship between Rb dephosphorylation and antiproliferative response suggests that early decreases in Rb phosphorylation could potentially identify patients with primary resistance. Kumarasamy et al. confirmed that activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity to CDK4/6 inhibition, with RB loss rendering cells completely independent of CDK4 and CDK6.

Ma et al. found that resistance to palbociclib was associated with persistent E2F-target gene expression, indicating ongoing Rb pathway activity despite CDK4/6 inhibition. Specifically, nonluminal subtypes demonstrated persistent elevation of CCND3, CCNE1, and CDKN2D, markers of continued E2F activity that bypass palbociclib’s mechanism.

Cell Cycle Checkpoint Proteins

The p27 protein emerged as a critical modulator of sensitivity. Kumarasamy et al. showed that protein levels of p27 were associated with cell cycle plasticity and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via SKP2 inhibition or MEK/ERK pathway targeting enhanced the cytostatic effect of CDK4/6 inhibitors. In ER+ xenograft models, few cells retained RB phosphorylation during palbociclib treatment, which was associated with limited p27 protein levels, suggesting that p27 levels influence the durability of palbociclib’s effects.

Downstream Signaling Pathways

Maskey et al. identified mTORC1 activity as a critical determinant of cell fate during CDK4/6 inhibition. In CAMA1 cells, mTORC1 activity remained elevated during palbociclib treatment, while in MCF7 and T47D cells, mTORC1 was suppressed. Importantly, inhibition of mTORC1 signaling via rapamycin or Raptor knockdown during palbociclib treatment blocked the induction of complete senescence in CAMA1 cells. Genetic depletion of TSC2, a negative regulator of mTORC1, resulted in sustained mTORC1 activity during palbociclib treatment and evoked a complete senescence response in MCF7 cells, demonstrating that persistent mTORC1 signaling can convert reversible to irreversible growth arrest.

Vijayaraghavan et al. discovered that autophagy is induced as a stress response to palbociclib. Palbociclib treatment decreased pRb and total Rb levels, but autophagy activation served as a resistance mechanism by degrading reactive oxygen species and potentially reversing G1 arrest. Combined inhibition of CDK4/6 and autophagy produced synergistic effects, suggesting that autophagy represents an adaptive survival pathway during cell cycle arrest.

Clinical Efficacy Outcomes

Clinical efficacy varied by disease setting, with the most robust data emerging from trials in hormone receptor-positive breast cancer.

Neoadjuvant Setting

Study

Clinical Response Rate

Pathological Response

Time-Based Outcomes

Maintenance of Arrest

Ma et al., 2017

80% (exam), 41% (ultrasound), 52% (mammogram)

No pathologic complete responses; significant reduction in tumor stages

Not mentioned

Continuous therapy necessary to maintain CCCA

Johnston et al., 2019

CR+PR: 54.3% (palbociclib+letrozole) vs 49.5% (letrozole); PD: 3.2% vs 5.4%

Not mentioned

Not mentioned

Not mentioned

Arnedos et al., 2018

Antiproliferative response: 58% (palbociclib) vs 12% (control); Ki67 decrease significant

Not mentioned

Not mentioned

Not mentioned

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In the neoadjuvant setting, palbociclib demonstrated robust antiproliferative activity but variable impact on tumor size reduction. Johnston et al. found that adding palbociclib to letrozole significantly enhanced Ki-67 suppression but did not increase clinical response rates over 14 weeks, possibly related to concurrent reduction in apoptosis. Ma et al. achieved an 80% clinical response rate by physical examination, though ultrasound and mammogram assessments showed lower rates (41% and 52%, respectively). No pathologic complete responses were observed, but significant reductions in tumor stage occurred.

Advanced Disease Setting

Study

CBR

PFS

Overall Survival

Response Duration

Turner et al., 2015

Not mentioned

Median 9.2 months (palbociclib+fulvestrant) vs 3.8 months (placebo+fulvestrant); HR 0.42

Not mentioned

Not mentioned

Vijayaraghavan et al., 2017

Not mentioned

Palbociclib doubles PFS vs letrozole or fulvestrant alone; Rb+/LMWE- tumors: median 436.5 months with letrozole, 10.7 months with fulvestrant

Not mentioned

Not mentioned

DeMichele et al., 2014

19% overall, 21% in HR+, 29% in HR+/HER2- with ≥2 prior endocrine therapies

Median 3.7 months overall

Not mentioned

Not mentioned

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The PALOMA-3 trial demonstrated that palbociclib combined with fulvestrant resulted in significantly longer progression-free survival (9.2 vs 3.8 months, HR 0.42) in patients with hormone receptor-positive metastatic breast cancer who had progressed on prior endocrine therapy. Vijayaraghavan et al. reported that palbociclib doubled PFS compared to endocrine therapy alone, with particularly prolonged PFS in Rb-positive, low-molecular-weight cyclin E (LMWE)-negative tumors (median 436.5 months with letrozole). DeMichele et al. observed modest clinical benefit rates of 19% overall, increasing to 29% in HR+/HER2- patients who had failed at least two prior endocrine therapies, with median PFS of 3.7 months.

Preclinical Efficacy

Asghar et al. demonstrated tumor reductions in 7 out of 10 mice treated with palbociclib in the LAR subtype of triple-negative breast cancer xenografts. Cell cycle length was prolonged in CDK2-high cells (20 hours vehicle vs 38 hours palbociclib), though cells showed adaptation to CDK4/6 inhibition over time.

Cellular Consequences and Senescence Phenotypes

Studies revealed heterogeneity in cellular responses to CDK4/6 inhibition, with some cell lines undergoing reversible arrest while others entered irreversible senescence.

Reversibility of Growth Arrest

Maskey et al. demonstrated that ER+ breast cancer cell lines exhibited distinct responses to palbociclib: MCF7 and T47D cells showed reversible G1-phase arrest with an incomplete senescence phenotype, whereas CAMA1 cells underwent irreversible cell cycle arrest and complete senescence. This difference correlated with mTORC1 signaling patterns, with sustained activity promoting complete senescence. Ma et al. confirmed the reversibility in clinical samples, showing that Ki67 levels rebounded at surgery after palbociclib discontinuation, though this rebound was suppressed by continuing palbociclib therapy.

Vijayaraghavan et al. found that reversibility was dose-dependent: low doses of palbociclib resulted in reversible G1 arrest, while higher doses led to irreversible growth inhibition. Palbociclib induced senescence markers including increased SA-β-gal activity and cellular complexity, but critically, did not induce apoptosis as evidenced by lack of cleaved PARP and caspase activity.

Autophagy and Survival Responses

Autophagy emerged as an important adaptive mechanism. Vijayaraghavan et al. showed that palbociclib induced autophagy as a stress response, and combining palbociclib with autophagy inhibitors like hydroxychloroquine (HCQ) led to sustained growth inhibition and irreversible senescence without inducing apoptosis. SA-β-gal staining confirmed senescence induction both in vitro and in vivo.

Apoptosis Markers

Johnston et al. observed suppression of cleaved PARP with palbociclib plus letrozole treatment, indicating reduced rather than increased apoptosis. The median log-fold suppression of cleaved PARP was greater with combination therapy (-0.80 vs -0.42). This concurrent reduction in both proliferation and apoptosis may explain why enhanced antiproliferative effects did not translate to proportional increases in clinical response rates.

Predictive Biomarkers and Resistance Mechanisms

Substantial heterogeneity in treatment response prompted investigation of predictive biomarkers and resistance pathways.

Rb Status as a Core Biomarker

Retinoblastoma status emerged as the most fundamental predictor of response. Ma et al. identified RB1 mutations, particularly frameshift mutations, as associated with resistance to palbociclib in HER2-enriched tumors, though missense RB1 mutations were found in tumors sensitive to treatment. Kumarasamy et al. confirmed that RB loss rendered cells completely independent of CDK4/6 activity, with RB phosphorylation status serving as a marker for treatment failure. Vijayaraghavan et al. demonstrated that Rb-positive status predicted sensitivity, with Rb-positive/LMWE-negative tumors showing the longest progression-free survival. Asghar et al. confirmed that loss of RB1 caused resistance to CDK4/6 inhibition through disruption of the CDK4/6-RB1 axis that controls the restriction point in G1 phase.

Cyclin E and CDK2 Activity

Cyclin E levels, particularly the low-molecular-weight isoform (LMWE), predicted resistance. Ma et al. found that CCNE1 gain was associated with resistance to CDK4/6 inhibition, likely through CDK2 activation. Vijayaraghavan et al. showed that Rb-positive but LMWE-positive tumors had reduced sensitivity, with overexpression of LMWE conferring resistance. Asghar et al. revealed that high cyclin E1 expression activated CDK2 and was dysregulated in resistant cells, with palbociclib-resistant basal-like TNBC cells exiting mitosis directly into a proliferative state with high CDK2 activity, bypassing the need for CDK4/6. Johnston et al. noted that response to palbociclib was correlated with RB1 mutation status but occurred independently of PIK3CA or PTEN mutations.

Molecular Subtypes

Ma et al. demonstrated that luminal subtypes (LumA and LumB) were more responsive to palbociclib, while nonluminal subtypes including basal-like and HER2-enriched tumors showed resistance. Persistent E2F-target gene expression, indicated by elevated CCND3, CCNE1, and CDKN2D, was linked to resistance. Asghar et al. found that the luminal androgen receptor (LAR) subtype of triple-negative breast cancer was highly sensitive to CDK4/6 inhibition, while basal-like subtypes were resistant.

Downstream Signaling Pathways

Kumarasamy et al. identified p27 protein levels as associated with cell cycle plasticity and sensitivity, with targeting of the MEK/ERK pathway and SKP2 inhibition emerging as strategies to enhance response. Maskey et al. showed that sustained mTORC1 activity during palbociclib treatment promoted complete senescence, suggesting mTORC1 inhibition via rapamycin or Raptor knockdown as a combination strategy.

Combination Strategies to Overcome Resistance

Several effective combinations emerged from these studies:

Safety and Tolerability Profile

Hematologic toxicities, particularly neutropenia, dominated the adverse event profile across clinical trials, though these were generally manageable with dose modifications.

Study

Grade 3/4 Neutropenia

Grade 3/4 Other Cytopenias

Dose Modifications

Non-Hematologic Toxicities

Ma et al., 2017

G3: 22%, G4: 4%

Leukopenia mentioned

14% required dose reductions

Fatigue, rash; no G4+ non-hematologic AEs

Johnston et al., 2019

Part of 49.8% G3+ toxicity

Asymptomatic neutropenia primary cause

21.6% interruptions/delays, 2.0% dose reductions

Not specified

Turner et al., 2015

62.0%

Leukopenia 25.2%, anemia 2.6%, thrombocytopenia 2.3%

Discontinuation: 2.6% palbociclib, 1.7% placebo

Fatigue 2.0%; febrile neutropenia 0.6%

Vijayaraghavan et al., 2017

56%

Leukopenia 25.2%

Higher doses (75-150 mg/kg) caused significant weight loss

Not specified; combination with HCQ well tolerated

DeMichele et al., 2014

51%

Anemia 5%, thrombocytopenia 22%

51% dose reductions, 24% interruptions

Not mentioned; cytopenias uncomplicated and easily managed

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Turner et al. reported the highest rate of grade 3/4 neutropenia at 62.0%, compared to 0.6% in the placebo group, along with leukopenia (25.2%), anemia (2.6%), and thrombocytopenia (2.3%). Critically, febrile neutropenia remained rare at 0.6% in both treatment arms, and discontinuation rates due to adverse events were low (2.6% with palbociclib vs 1.7% with placebo). Johnston et al. found that 49.8% of patients experienced grade 3 or greater toxicity with palbociclib plus letrozole versus 17.0% with letrozole alone, primarily driven by asymptomatic neutropenia, with treatment interruptions or delays in 21.6% and dose reductions in only 2.0%.

DeMichele et al. emphasized that cytopenias were uncomplicated and easily managed with dose reduction, with 51% of patients requiring dose modifications and 24% experiencing treatment interruptions. Ma et al. observed grade 3 neutropenia in 22% and grade 4 in 4% of patients, with 14% requiring dose reductions due to neutropenia, elevated transaminases, or rash. No grade 4 or higher non-hematologic adverse events occurred.

Vijayaraghavan et al. noted in preclinical models that optimization of palbociclib dosing was crucial, as higher doses (75 or 150 mg/kg) caused significant body weight loss, but combination with hydroxychloroquine was well tolerated without changes in body weight or blood counts.

Synthesis

The body of evidence reveals consistent mechanisms but variable clinical outcomes that require reconciliation. While palbociclib uniformly induced G1 cell cycle arrest across studies, the durability of this arrest and translation to clinical benefit varied substantially based on molecular context, treatment duration, and combination strategies.

Context-Dependent Response Patterns

The heterogeneity in clinical benefit rates—ranging from 19% in heavily pretreated advanced disease to 87% complete cell cycle arrest in neoadjuvant settings—reflects differences in disease burden, prior treatment exposure, and measurement endpoints rather than conflicting results. In the neoadjuvant setting with less advanced disease, both Ma et al. and Johnston et al. achieved >85% complete cell cycle arrest rates, demonstrating maximal antiproliferative activity when palbociclib is combined with endocrine therapy in treatment-naive tumors. Conversely, DeMichele et al.’s lower clinical benefit rate of 19% occurred in patients with a median of 2 prior cytotoxic regimens, suggesting that heavily pretreated tumors may harbor additional resistance mechanisms beyond those addressed by CDK4/6 inhibition alone.

The progression-free survival benefit also follows a dose-response pattern by line of therapy. Turner et al. achieved 9.2 months median PFS in endocrine-resistant disease, while Vijayaraghavan et al. reported median PFS exceeding 400 months in optimal biomarker-selected populations (Rb+/LMWE-). Both findings are valid within their respective contexts: the former represents a clinically heterogeneous population without biomarker selection, while the latter represents a molecularly defined subset with intact G1/S checkpoint machinery.

Mechanisms Explaining Divergent Cellular Responses

The contrast between reversible and irreversible growth arrest observed by Maskey et al. and Vijayaraghavan et al. can be mechanistically explained through mTORC1 activity and autophagy induction. CAMA1 cells maintained elevated mTORC1 signaling during palbociclib treatment, leading to irreversible senescence, whereas MCF7 and T47D cells suppressed mTORC1 and experienced reversible arrest. This divergence is not contradictory but rather demonstrates that cellular context—specifically basal mTORC1 activity and the cell’s ability to modulate this pathway—determines whether CDK4/6 inhibition causes cytostatic or senescent outcomes. The finding that TSC2 depletion converted MCF7 cells from reversible to irreversible arrest provides direct mechanistic evidence that sustained mTORC1 activity is sufficient to drive complete senescence.

Similarly, Vijayaraghavan et al.’s observation that autophagy serves as an adaptive resistance mechanism explains why some cells escape permanent growth inhibition. Autophagy activation allows cells to survive metabolic stress during G1 arrest, potentially explaining the reversibility observed in certain contexts. The synergy between palbociclib and autophagy inhibitors validates that blocking this survival pathway converts cytostatic arrest into terminal senescence.

Biomarker-Driven Sensitivity Predictions

The apparent contradiction between studies reporting universal benefit and those identifying biomarker-dependent responses reflects measurement granularity rather than conflicting findings. While Turner et al. found consistent benefits across subgroups in their phase 3 trial, this population-level observation does not negate the molecular determinants identified in more detailed mechanistic studies.

Ma et al., Vijayaraghavan et al., and Asghar et al. converge on a unified model: intact Rb and absent or low cyclin E (particularly LMWE) predict maximal sensitivity, while RB1 loss or LMWE overexpression confers resistance. Kumarasamy et al.’s identification of p27 levels as modulators of sensitivity adds nuance to this model—p27 acts as a rheostat determining the threshold at which cells commit to arrest versus escaping through CDK2 activation. Asghar et al.’s single-cell analysis revealed that sensitive LAR cells exit mitosis with low CDK2 activity and require CDK4/6 for reentry, whereas resistant basal-like cells maintain high CDK2 activity post-mitosis, bypassing the CDK4/6 requirement. Both phenotypes coexist within TNBC but respond differently based on their intrinsic cell cycle dynamics.

Non-Linear Relationships: Proliferation vs. Clinical Response

Johnston et al.’s finding that enhanced Ki-67 suppression did not translate to increased clinical response rates initially appears contradictory but is explained by the concurrent suppression of apoptosis. The reduction in cleaved PARP indicates that while palbociclib profoundly arrests proliferation, it simultaneously protects cells from apoptotic death. This creates a cytostatic rather than cytotoxic effect, where tumor cells remain viable but non-proliferative. Over the 14-week treatment period, this translates to disease stabilization rather than tumor regression, explaining why complete cell cycle arrest rates of 90% do not correspond to 90% clinical responses.

This cytostatic mechanism also explains Ma et al.’s observation that continuous therapy is necessary to maintain antiproliferative effects. When palbociclib is discontinued, arrested cells with intact Rb and no terminal senescence can resume cycling once CDK4/6 activity is restored. The addition of cycle 5 palbociclib immediately before surgery suppressed Ki67 rebound, demonstrating that the arrest is maintained only through continuous CDK4/6 inhibition in populations that have not undergone irreversible senescence.

Combination Strategies Addressing Distinct Resistance Nodes

The diverse combination strategies emerging from these studies—autophagy inhibition, PI3K inhibition, MEK inhibition, and mTORC1 modulation—target mechanistically distinct resistance pathways that become activated during G1 arrest. Autophagy inhibition blocks stress-induced survival responses, PI3K inhibition addresses PIK3CA-mutant tumors where constitutive signaling bypasses G1 arrest requirements, and MEK inhibition upregulates p27 to lower the threshold for CDK4/6 dependence. These are not competing strategies but complementary approaches applicable to different molecular contexts.

The MEK combination is particularly rational given Kumarasamy et al.’s demonstration that p27 induction via MEK/ERK pathway inhibition enhances palbociclib efficacy. This mechanistically addresses the subset of tumors with low basal p27 that maintain cell cycle plasticity. Similarly, Tien & Sadar’s sequential dosing strategy in androgen receptor-positive cancers recognizes that different agents target different cell cycle phases—palbociclib delays G1-S transition while EPI-7170 targets S-phase cells—and sequential administration maximizes the proportion of cells arrested in each vulnerable phase.

Quality Hierarchy Considerations

The phase 3 PALOMA-3 trial by Turner et al. carries the greatest weight for establishing clinical efficacy in advanced HR+ breast cancer, with its randomized design, large sample size (n=521), and definitive progression-free survival benefit (HR 0.42). The neoadjuvant trials by Ma et al. and Johnston et al., though smaller phase 2 studies, provide complementary mechanistic insights through serial biopsies that were not feasible in the advanced disease setting. The preclinical studies by Maskey et al., Vijayaraghavan et al., Asghar et al., and Kumarasamy et al. elucidate mechanisms that inform biomarker development but require clinical validation. The convergence of clinical efficacy data from Turner et al. with mechanistic predictions from preclinical models (particularly regarding Rb status) strengthens confidence in these biomarkers despite coming from studies of varying design rigor.

Resolved Model of Palbociclib Action

Integrating these findings yields a coherent model: palbociclib reliably induces G1 arrest in Rb-proficient cells by preventing Rb hyperphosphorylation and maintaining E2F target suppression. The durability of this arrest depends on three factors: (1) CDK2 activity levels, with high CDK2/cyclin E bypassing the G1 block; (2) mTORC1 activity during arrest, with sustained signaling driving irreversible senescence and suppressed signaling allowing reversible arrest; and (3) autophagy induction as a survival response that can be targeted for combination therapy. Clinical benefit requires both achieving cell cycle arrest and maintaining it, explaining why continuous dosing is necessary and why biomarker selection (Rb+/LMWE-/adequate p27) identifies patients with maximal benefit.

References

Reeja S. Maskey, F. Wang, Elyssa Lehman, Yiqun Wang, N. Emmanuel, and 6 more\ (2020).Sustained mTORC1 activity during palbociclib-induced growth arrest triggers senescence in ER+ breast cancer cells. Cell Cycle

Cynthia X. Ma, F. Gao, Jingqin R Luo, D. Northfelt, M. Goetz, and 29 more\ (2017).NeoPalAna: Neoadjuvant Palbociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, and Anastrozole for Clinical Stage 2 or 3 Estrogen Receptor–Positive Breast Cancer. Clinical Cancer Research

N. Turner, J. Ro, F. André, S. Loi, S. Verma, and 9 more\ (2015).Palbociclib in Hormone-Receptor-Positive Advanced Breast Cancer. New England Journal of Medicine

S. Vijayaraghavan, Cansu Karakas, I. Doostan, Xian Chen, Tuyen N Bui, and 12 more\ (2017).CDK4/6 and autophagy inhibitors synergistically induce senescence in Rb positive cytoplasmic cyclin E negative cancers. Nature Communications

A. DeMichele, A. Clark, K. Tan, D. Heitjan, Kristi Gramlich, and 13 more\ (2014).CDK 4/6 Inhibitor Palbociclib (PD0332991) in Rb+ Advanced Breast Cancer: Phase II Activity, Safety, and Predictive Biomarker Assessment. Clinical Cancer Research

A. Tien, M. Sadar\ (2021).Cyclin-dependent Kinase 4/6 Inhibitor Palbociclib in Combination with Ralaniten Analogs for the Treatment of Androgen Receptor–positive Prostate and Breast Cancers. Molecular Cancer Therapeutics

U. Asghar, A. Barr, R. Cutts, M. Beaney, I. Babina, and 9 more\ (2017).Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer. Clinical Cancer Research

V. Kumarasamy, Paris Vail, Ram Nambiar, A. Witkiewicz, E. Knudsen\ (2020).Functional Determinants of Cell Cycle Plasticity and Sensitivity to CDK4/6 Inhibition. Cancer Research

S. Johnston, S. Puhalla, D. Wheatley, A. Ring, P. Barry, and 32 more\ (2019).Randomized Phase II Study Evaluating Palbociclib in Addition to Letrozole as Neoadjuvant Therapy in Estrogen Receptor-Positive Early Breast Cancer: PALLET Trial. Journal of Clinical Oncology

M. Arnedos, M. Bayar, B. Cheaib, Véronique Scott, I. Bouakka, and 14 more\ (2018).Modulation of Rb phosphorylation and antiproliferative response to palbociclib: the preoperative-palbociclib (POP) randomized clinical trial. Annals of Oncology

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NeoPalAna: Neoadjuvant Palbociclib, a Cyclin-Dependent Kinase 4/6 Inhibitor, and Anastrozole for Clinical Stage 2 or 3 Estrogen Receptor–Positive Breast Cancer

Cynthia X. Ma, F. Gao, Jingqin R Luo, D. Northfelt, M. Goetz, A. Forero, J. Hoog, M. Naughton, F. Ademuyiwa, R. Suresh, K. Anderson, J. Margenthaler, R. Aft, T. Hobday, T. Moynihan, W. Gillanders, Amy E. Cyr, T. Eberlein, T. Hieken, H. Krontiras, Zhanfang Guo, Michelle V Lee, N. Spies, Zachary L. Skidmore, O. Griffith, M. Griffith, S. Thomas, Caroline Bumb, Kiran R Vij, C. H. Bartlett, M. Koehler, H. Al-Kateb, Souzan Sanati, M. Ellis

Clinical Cancer Research·

2017·

287 citations

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Study Design

- Study type: Single-arm phase II neoadjuvant clinical trial - Setting: Patients with clinical stage II/III ER+/HER2− breast cancer - Cancer type and subtype: ER+/HER2− breast cancer - Sample size and key patient characteristics: 50 patients (18 pre- and 32 post-menopausal), median age 57.5 years, clinical stage II-III ER+/HER2− invasive breast cancer

CDK4/6 Inhibitor Details

- Specific CDK4/6 inhibitor used: Palbociclib - Dose and schedule: 125 mg daily on days 1–21 of each 28-day cycle - Duration of treatment: Four 28-day cycles, with some patients receiving an additional 10 to 12 days (Cycle 5) before surgery - Combination therapies: Anastrozole (1 mg daily) and goserelin (if premenopausal) - Administration route and timing: Oral (PO) administration

Cell Cycle Arrest Measurements

- Ki-67 levels: Baseline (C0D1), post-anastrozole (C1D1), post-palbociclib (C1D15) - Complete cell cycle arrest rates: 26% at C1D1, 87% at C1D15 - Proliferation markers: PAM50 11-gene proliferation score - Time points measured: Baseline (C0D1), C1D1, C1D15, surgery

Mechanism Analysis

- Rb pathway activity: Palbociclib inhibits CDK4/6, which phosphorylates Rb, leading to E2F release and cell proliferation. Resistance is associated with persistent E2F-target gene expression. - Cyclin D1/CDK4/CDK6 expression and activity: Palbociclib is a potent CDK4/6 inhibitor, enhancing cell cycle control over anastrozole. - Cell cycle checkpoint proteins: Not explicitly mentioned in the context of CDK4/6 inhibition. - Downstream signaling pathways: Not explicitly mentioned in the context of CDK4/6 inhibition. - Molecular targets and biomarkers studied: RB1 status and E2F-target gene expression are important for resistance.

Clinical Outcomes

- Response rates: 80% (exam), 41% (ultrasound), 52% (mammogram) - Clinical benefit rate: Not explicitly mentioned - Pathological response: No pathologic complete responses; significant reduction in tumor stages - Time to progression and overall survival: Not mentioned - Response duration and maintenance of cell cycle arrest: Continuous therapy with palbociclib necessary to maintain CCCA

Senescence and Cell Fate

- Senescence markers and phenotype: Not mentioned - Reversibility vs irreversibility of arrest: Reversible; Ki67 levels rebounded at surgery after palbociclib was stopped, but this rebound was suppressed by continuing palbociclib. - Apoptosis markers: Not mentioned - Autophagy activation: Not mentioned - Long-term cell fate outcomes: Not mentioned

Resistance and Predictive Factors

- Predictive biomarkers: RB1 status (mutations associated with resistance), cyclin E levels (CCNE1 gain associated with resistance) - Resistance mechanisms and pathways: Persistent E2F-target gene expression, CCND3, CCNE1, and CDKN2D elevation - Factors associated with treatment failure: Nonluminal subtypes (basal-like, HER2-E), RB1 mutations - Molecular subtypes or patient characteristics affecting response: Luminal subtypes (LumA, LumB) are more responsive; nonluminal subtypes are less responsive - Combination strategies to overcome resistance: Sequential treatment approach for individualized response assessment and treatment recommendations

Toxicity Profile

- Grade 3/4 adverse events: Transient neutropenia (G3, 22%; G4, 4%), leukopenia, fatigue. - Dose modifications: 14% of patients required dose reductions due to G3 neutropenia, G4 neutropenia, G3 elevated transaminases, and G2 rash. - Cytopenias: Neutropenia (G3, 22%; G4, 4%); no mention of anemia or thrombocytopenia. - Non-hematologic toxicities: Fatigue, rash; no G4 or above non-hematologic AEs. - Management strategies: Dose reductions and delays.

Purpose: Cyclin-dependent kinase (CDK) 4/6 drives cell proliferation in estrogen receptor–positive (ER+) breast cancer. This single-arm phase II neoadjuvant trial (NeoPalAna) assessed the antiproliferative activity of the CDK4/6 inhibitor palbociclib in primary breast cancer as a prelude to adjuvant studies. Experimental Design: Eligible patients with clinical stage II/III ER+/HER2− breast cancer received anastrozole 1 mg daily for 4 weeks (cycle 0; with goserelin if premenopausal), followed by adding palbociclib (125 mg daily on days 1–21) on cycle 1 day 1 (C1D1) for four 28-day cycles unless C1D15 Ki67 > 10%, in which case patients went off study due to inadequate response. Anastrozole was continued until surgery, which occurred 3 to 5 weeks after palbociclib exposure. Later patients received additional 10 to 12 days of palbociclib (Cycle 5) immediately before surgery. Serial biopsies at baseline, C1D1, C1D15, and surgery were analyzed for Ki67, gene expression, and mutation profiles. The primary endpoint was complete cell cycle arrest (CCCA: central Ki67 ≤ 2.7%). Results: Fifty patients enrolled. The CCCA rate was significantly higher after adding palbociclib to anastrozole (C1D15 87% vs. C1D1 26%, P < 0.001). Palbociclib enhanced cell-cycle control over anastrozole monotherapy regardless of luminal subtype (A vs. B) and PIK3CA status with activity observed across a broad range of clinicopathologic and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with nonluminal subtypes and persistent E2F-target gene expression. Conclusions: Palbociclib is an active antiproliferative agent for early-stage breast cancer resistant to anastrozole; however, prolonged administration may be necessary to maintain its effect. Clin Cancer Res; 23(15); 4055–65. ©2017 AACR.

Introduction

The cyclin-dependent kinase (CDK) 4/6 in association with D-type cyclins promotes G1/S phase transition through phosphorylation of the retinoblastoma susceptibility (RB1) gene product (Rb) and other members of the pocket protein family (p107 and p130) (1)(2)(3)(4). Hyperphosphorylated Rb releases E2F and DP transcription factors, which activate the expression of genes required for cell proliferation (5). As cyclin D1 is a direct transcriptional target of estrogen receptor (ER), there is a direct association between ER signaling and CDK4/6 activation (6)(7)(8)(9). In addition, estrogen-independent CDK4/6 activation occurs as a result of other mitogenic signaling or genomic alterations, leading to endocrine resistance (10).

Palbociclib is a potent selective inhibitor of CDK4/6 (11) which exerts synergistic antitumor effect when combined with endocrine therapy in both endocrine sensitive and resistant luminal breast cancers (12). The addition of palbociclib to endocrine therapy significantly improved progression free survival (PFS) in patients with hormone receptor positive, HER2 negative metastatic breast cancer (13)(14)(15)(16). The effect of palbociclib in combination with endocrine therapy in early stage disease had not been determined. We therefore conducted a neoadjuvant phase II trial (NeoPalAna, Clinical Trials.gov#: NCT01723774) to determine the anti-proliferative activity of palbociclib when added to anastrozole in patients with newly diagnosed clinical stage II/III ER+/HER2-breast cancer as a prelude to adjuvant studies and to discover predictive biomarkers potentially useful for defining the appropriate adjuvant population.

The primary objective was to determine whether the addition of anastrozole to palbociclib induces a higher rate of Complete Cell Cycle Arrest (CCCA: Ki67≤2.7%) than that achieved by anastrozole alone administered as initial therapy. The primary endpoint was chosen based on the long-term follow-up data of several neoadjuvant endocrine therapy trials which demonstrated that the 2.7% Ki67 cut-point (natural log of one) during neoadjuvant endocrine treatment is associated with favorable breast cancer relapse free and overall survival (17,18). While this single arm study was designed based on the rates of CCCA observed in previous neoadjuvant aromatase inhibitor (AI) studies the added effect of palbociclib over that of anastrozole was determined by analysis of tumor biopsies collected at C1D1 following 4 weeks of cycle 0 anastrozole monotherapy, and at C1D15, 2 weeks post the addition of palbociclib to anastrozole. CCCA and Ki67 response were assessed by PIK3CA mutation status because of the alternative strategy of PIK3CA targeted therapy in the mutation positive population. Secondary objectives included analysis of CCCA and Ki67 response by baseline PAM50-based intrinsic subtypes, and assessment of clinical, radiological and pathological response and safety profiles. Exploratory biomarker studies included gene expression and somatic mutation profiling.

Eligibility

Eligible patients included pre-and post-menopausal women at least 18 years old, with a clinical stage II-III, ER+ (Allred score 6-8) and HER2-(0 or 1+ by IHC or FISH negative) invasive breast cancer. Additional eligibility criteria included: Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) 0-2, adequate organ and marrow function. For patients receiving goserelin, estradiol level in the postmenopausal range was required to receive further treatment on study. Exclusion criteria included prior treatment of the current cancer, uncontrolled intercurrent illness, active or recent coronary events, cerebrovascular accident, symptomatic pulmonary embolism or congestive heart failure, known HIVpositivity, metastatic disease, inflammatory cancer, previous excisional biopsy of the breast or sentinel lymph node, corrected QT>470msec, allergic reactions to compounds similar to palbociclib, pregnant/nursing, or taking anticoagulation, medications that prolong QT or are known CYP3A4 inhibitors. The study was approved by Institutional Review Board at participating sites and followed the Declaration of Helsinki and Good Clinical Practice guidelines. Written informed consent was required.

Study Design and Treatment

The primary endpoint was CCCA (Ki67≤ 2.7%) on palbociclib plus anastrozole at C1D15. The study was designed to ensure the sample size for the PIK3CA WT cohort and the overall population for the primary endpoint analysis. A sample size of 33 in the PIK3CA WT cohort was chosen based on the Fleming's single-stage phase II design to test the hypothesis that palbociclib plus anastrozole leads to at least 50% improvement over anastrozole alone in CCCA rates (44% with anastrozole based on historical data (19), vs 66% with palbociclib plus anastrozole, power=0.8, alpha=0.05). The primary endpoint is met if more than 20/33 patients achieved CCCA. Patients were prospectively assigned to PIK3CA WT or Mut Cohort at C1D1 based on CLIA PIK3CA sequencing. Based on the prevalence of PIK3CA mutation, we estimated that 14-17 patients would enroll to the exploratory PIK3CA Mut cohort with 33 patients to the PIK3CA WT cohort. If 10 of 15 achieved CCCA in the PIK3CA Mut cohort, the 80% confidence for the "true" rate would be 47%-83%.

Eligible patients were pre-registered, underwent baseline tumor biopsy (C0D1) and began cycle 0 anastrozole (1mg PO daily for 4 weeks) and goserelin (3.6mg SC each 28 days) if premenopausal, while PIK3CA sequencing was being performed. Palbociclib (125mg PO daily on D1-21 each 28-day cycle) was started on C1D1 after tumor biopsy (2 nd biopsy time-point) and registration to PIK3CA WT or Mut Cohort. Patients with unsuccessful PIK3CA sequencing due to DNA quality or quantity not sufficient (QNS) also received therapy per protocol. Tumor biopsy was again performed on C1D15 (3 rd biopsy time-point) for CLIA Ki67 analysis. If C1D15 Ki67>10%, protocol therapy was discontinued due to inadequate response. Patients with C1D15 Ki67≤10% (or indeterminant) continued palbociclib and anastrozole for 4 cycles unless patients experienced intolerable side effects, disease progression, estradiol level in premenopausal range while receiving goserelin, or withdrew. Surgery occurred 3-5 weeks post the last dose of palbociclib to allow adverse event (AE) recovery. Following a protocol amendment, patients whose ANC recovered to >1.5k/mcL, platelet >100k/mcL and non-hematologic AEs to ≤grade 1 within 3 weeks after completion of cycle 4 received additional 10-12 days of cycle 5 palbociclib immediately before surgery. Anastrozole (with goserelin if premenopausal) was administered until surgery. Uniform biopsy/shipment kits were provided (18,19).

Clinical tape/caliper bi-dimensional tumor measurement and AE assessment by CTCAE 4.0 were performed on day 1 of each cycle. Serum estradiol levels were tested on C1D1 and C3D1. Radiological assessment by mammogram and ultrasound was required at baseline and prior to surgery. Clinical tumor response was assessed by WHO criteria and radiologic response by RECIST 1.1.

CLIA PIK3CA Sequencing

PIK3CA (ref seq# NM_006218) sequencing of exons 2, 5, 8, 10 and 21, and exon-intron splice junctions was performed on tumor DNA from the baseline biopsy at the CLIA certified Washington University Genomic and Pathology Service initially by Sanger (n=18), subsequently by next-generation sequencing (NGS) (n=32). The targeted exons were PCR amplified using the (Fluidigm Corp., South San Francisco, USA) 48.48 high-throughput access array system. Cluster generation and sequencing was performed using Illumina's HiSeq2500 Reagent Kit (200 cycles) and 2x101 paired-end sequence reads were generated. Each patient's tumor DNA was processed and sequenced in three independent technical replicates. The three fastq files were each aligned independently to the human reference genome hg19 NCBI build 37.2 to generate three BAM files, then merged to a single BAM file. Sequence analysis was performed on all four BAM files using a combination of commercially available and custom developed scripts to generate a multisample VCF file. Alignment to the human reference genome was performed using Novoalign 2.08.02, sorting and indexing was performed by SAMtools 0.1.18-1, with coverage calculation by BedTools 2.13.3 and variants calling by Freebayes 0.9.7. Variants were called if present in at least three of the four BAM files and the average variant allele frequency (VAF) ≥10%.

Ki67 immunohistochemistry (IHC) and Quantification

Ki67 staining was performed centrally at the CAP/CLIA certified AMP lab at Washington University using the CONFIRM anti-Ki67 antibody (clone 30-9) and scored using pathologist-guided imaging analysis as previously described (18,19).

Gene expression analysis, PAM50 intrinsic subtype and proliferation score

Total RNA from fresh-frozen tumor biopsies at baseline and subsequent time-points was extracted when at least 50% tumor cellularity was present (19). Microarray gene expression data were generated on an Agilent microarray platform (Santa Clara, CA) and normalized (19), followed by PAM50-based intrinsic subtype assignment and the 11-gene proliferation score determination (19,20).

83-gene panel next generation sequencing

Tumor DNA extracted from fresh frozen biopsies and matched leukocyte germline DNA were subjected to targeted Illumina NGS of an 83-gene panel (21)(22)(23). Mutation waterfall plot was created with GenVisr (24).

Statistical Analysis

The rates of CCCA (overall and by subgroups) were the percentage of patients with tumor Ki67≤2.7% at C1D1 and C1D15. The corresponding 90% confidence intervals (CI) were calculated as normal approximation or binomial exact CI as appropriate. The rates of CCCA between C1D1 and C1D15 were compared using McNemar test. The clinical response rate was the percentage of evaluable patients met the WHO criteria of complete or partial response prior to surgery with 90%CI. The radiological response rate (by RECIST1.1) was similarly calculated.

Changes in Ki67 over time and the differences between subgroups (PIK3CA Mut vs. WT, LumA vs. LumB subtypes, etc.) were analyzed using generalized estimating equation (GEE) (25,26), followed by a step-down Bonferroni adjustment for multiple comparisons. As Ki67 followed a right-skewed distribution, a logarithm transformation was performed. (25,26) The gene expression data were analyzed to identify differentially expressed genes across timepoints (by F-test) and between time-points (by moderated two sample t-test) accounting for multiple measures from the same patient using the Bioconductor package limma (linear models for microarray data, version 3.20.9) and false discovery rate adjusted P-values were reported (27). Enriched gene ontology terms were subsequently performed using GOstats (version 2.30.0) (28). All statistical analyses were performed using SAS 9.2 (SAS Institutes, Cary, NC) except gene expression analyses which were performed using R 3.1.1 (http:// cran.r-project.org). All reported P-values were two-sided unless otherwise noted.

Enrollment

Between April 2013 and April 2015, 50 patients (18 pre-and 32 post-menopausal), median age 57.5 (range 34.1-79.6) years, with clinical stage II-III ER+/HER2-breast cancer enrolled to the study, which included 16 in the PIK3CA Mut, 32 in the PIK3CA WT cohort and 2 with unknown PIK3CA status. Table 1 detailed the patient and tumor characteristics.

Five patients were without C1D15 Ki67 value due to withdrawal (n=2), inability to biopsy (n=2), and insufficient biopsy material (n=1), leaving 45 (16 PIK3CA Mut, 28 PIK3CA WT, and 1 PIK3CA unknown) evaluable for the primary endpoint (Supplementary Fig. S1 ). Five patients went off study per protocol due to C1D15 Ki67>10% (n=4) and elevated estradiol on goserelin at C3D1 (62 pg/ml) (n=1). Thirty-nine patients completed neoadjuvant therapy and underwent definitive breast and axillary surgery. Following the protocol amendment that added cycle 5 prior to surgery, 10 patients received cycle 5 palbociclib and all underwent surgery as scheduled. Among these 10 patients, 1 mistakenly stopped her palbociclib early (5 days prior to surgery) despite normal ANC 1 week on cycle 5, 1 had a planned 1-week delay in her surgery from the final dose of palbociclib due to physician concern of potential cytopenia since this patient experienced grade 3 ANC in previous cycles that led to a dose delay in cycle 4. Seven additional patients who would have been eligible but did not proceed with cycle 5 due to scheduling and logistic reasons (n=6), and ANC not recovering to normal within 3 weeks post cycle 4 (n=1).

Safety

All patients were evaluable for AE. Treatment was well tolerated. Common grade (G)2 and above AEs (>10% incidence) included transient neutropenia (G3, 22%; G4, 4%), leukopenia, and fatigue (Supplementary Table S1 ). Seven (14%) patients required one doselevel reduction due to G3 neutropenia with dose delay (n=3), G4 neutropenia (n=1), G3 elevated transaminases (n=2), and G2 rash (n=1). No G4 or above non-hematologic AEs or neutropenic fevers were observed.

Clinical, radiologic, and pathologic responses

Forty-one patients received at least 3 cycles of palbociclib and anastrozole, including: 39 underwent surgery, 1 refused surgery, and 1 subsequently withdrew, were assessed for clinical and radiologic responses (Supplementary Table S2 ). The response rates (90%CI) were 80%(68-90%), 41%(25-58%), and 52%(35-68%), by exam, ultrasound, and mammogram. Pathologic stages were: I (n=7), II (n=22), and III (n=10) at mastectomy (n=20) or lumpectomy (n=19). No pathologic complete responses were observed.

CCCA and Ki67 response in the overall population and by PIK3CA status

The rates of CCCA with palbociclib plus anastrozole were significantly higher at C1D15 than that at C1D1 with anastrozole monotherapy for all evaluable patients (87% vs 26%, p<0.001), PIK3CA Mut (100% vs 25%, p<0.001), and PIK3CA WT (79% vs 25%, p<0.001) cohort (Table 2 ). The CCCA rates at C1D15 exceeded the pre-defined cut-point for meeting the primary endpoint of at least 66% in the overall, PIK3CA WT and Mut cohorts. Of the 31 patients resistant to anastrozole (non-CCCA at C1D1), 26 (84%) responded to palbociclib (CCCA at C1D15). When considered as a continuous variable Ki67 levels were significantly reduced from baseline C0D1 to C1D1 following anastrozole monotherapy (p<0.01) and from C1D1 to C1D15 post adding palbociclib (p<0.01) for both PIK3CA WT and Mut cohorts (Fig. 1A-C ). There was a greater variability in Ki67 response among the PIK3CA WT tumors, which included all 6 palbociclib resistant tumors (non-CCCA at C1D15) (Fig. 1C ).

CCCA and Ki67 response by intrinsic subtype

Thirty-two patients had sufficient tumor RNA extracted from frozen baseline biopsies for microarray gene expression analysis. PAM50 intrinsic subtype determination identified 17 Luminal A (LumA), 12 Luminal B (LumB), 1 basal-like, 1 HER2-Enriched (HER2-E) (but HER2 negative by clinical criteria) and 1 normal-like assignment (Table 1 ). Significantly higher rates of CCCA were achieved at C1D15 compared to C1D1 in LumA (100% vs 40%, p=0.008) and LumB (75% vs 9%, p=0.02) tumors (Table 2 ). 17/21 (81%) (9/9 LumA, 8/10 LumB, and 0/2 non-luminal) tumors resistant to anastrozole subsequently achieved CCCA at C1D15. The subtypes of the six tumors resistant to palbociclib (C1D15 Ki67>2.7%) included 3 LumB, both non-luminal (1 basal-like, 1 HER2-E) and 1 subtype unknown (Supplementary Fig. S2 ). When considering Ki67 as a continuous variable, LumB tumors had significantly higher levels of Ki67 compared to Lum A tumors at C0D1 (p<0.01). In addition, Ki67 levels were significantly reduced from baseline (C0D1) to C1D1 by anastrozole monotherapy (p<0.01), and from C1D1 to C1D15 by the addition of palbociclib (p<0.01) in both LumA and LumB tumors (Fig. 1D-F ), indicating the efficacy of anastrozole and palbociclib in both luminal subtypes. Interestingly, the degree of Ki67 suppression by anastrozole (p=0.69) and by the addition of palbociclib (p=0.97) were similar between LumA and LumB tumors.

Ki67 recovery at surgery after palbociclib withdrawal

The first 29 patients completed 4 cycles of anastrozole plus palbociclib and underwent surgery following a median washout period of 29 (ranges 8-49) days from C4D21 palbociclib. Anastrozole was continued until surgery. Ki67 was significantly higher at surgery than C1D15 (p<0.01, n=23 paired samples), but not significantly different from C1D1 (p=0.077, adjusted for multiple comparisons) (Fig. 1G-H ). To test whether the Ki67 recovery was due to palbociclib withdrawal, subsequent patients (n=8) received additional 10-12 days of palbociclib immediately before surgery (cycle 5). There was no significant differences in Ki67 levels between surgery and C1D15 timepionts in these patients (p=0.68, n=7 paired samples) (Fig. 1G, I ). Six of the 7 cases remained in CCCA at surgery (Fig. 1I ), indicating the need for continuous therapy with palbociclib to maintain the anti-proliferative effect.

Ki67 response by clinical, pathological, and mutation profiles

To identify potential clinical and molecular response markers for palbociclib, we examined the rates of CCCA at C1D1 and C1D15 by menopausal status, histology, tumor grade, PgR status and mutations identified in the 83-gene panel NGS (Supplementary Table S3 ). Palbociclib benefit was observed across all subsets, including tumors that were grade 3, negative for PgR, or harboring mutations in TP53 or PTEN, which are known endocrine resistant mechanisms (Table 2 ). RB1 mutation was identified in 3 breast cancers, all with cooccurring PTEN mutations, at baseline (Fig. 2 ), including the RB1 E323fs (VAF 11% at baseline, 25% at C1D1) mutant HER2-E tumor that was resistant to both anastrozole and palbociclib, and two other tumors resistant to anastrozole alone (C1D1 Ki67>2.7%) but sensitive to palbociclib (C1D15 Ki67≦2.7%): one LumB tumor with RB1 I532N (VAF 5.4% at baseline, undetectable at C1D1, C1D15 and surgery) and the other (subtype unknown) carrying concurrent RB1 A562P (VAF 4.7%)/S576* (VAF 6.8%) at baseline (no mutation data for subsequent time-points). Fig. 2 details the somatic mutations available for 41 patients with anastrozole-sensitive (C1D1 Ki67≤2.7%; n=9), palbociclib-sensitive (C1D1 Ki67>2.7% but C1D15 Ki67≤2.7%; n=24), or resistant (C1D15 Ki67>2.7%, n=5) tumors (C1D1/C1D15 Ki67 missing, n=3). In addition to PIK3CA, mutations in CDH1, PTEN, TP53, TBX3 and MAP3K1 were most common. Five of the 6 resistant tumors had sufficient material for sequencing. The HER2-E tumor carried an RB1 pE323fs as discussed above, while the basal-like tumor harbored a TP53 p.S127F mutation. The 3 LumB resistant tumors carried TP53 T18fs (n=1), none (n=1), or mutations in multiple genes (ATR, MAP3K1, GATA3, MLL, BRCA2, FOXA1, AKT1 and CDH1) (n=1). Overall, palbociclib was effective across tumors harboring a wide spectrum of somatic mutations.

Response by PAM50 proliferation score

To confirm the added anti-proliferative effect of palbociclib over anastrozole based on Ki67 IHC, we also calculated the PAM50 proliferation score using the previously described 11gene signature (20,29) based on microarray data from each time-point. The proliferation score was significantly reduced from baseline to C1D1 (p<0.0001), and from C1D1 to C1D15 (p<0.0001) (Supplementary Fig. S3 ) and correlated with Ki67 data at all time-points (Supplementary Fig. S3B-E ). A heat-map of the 11 proliferation genes in different response categories is shown in Supplementary Fig. S4 . Similar to Ki67, recovery in the proliferation score was also observed at surgery, which was inhibited by cycle 5 palbociclib. This data provided further validation of the anti-proliferative effects of palbociclib.

Gene expression changes induced by anastrozole alone, and in combination with palbociclib

Agilent microarray for gene expression was performed using total RNA obtained from serial fresh frozen tumor biopsies. Data was generated for 29,284 probes and 118 samples (baseline, n=32; C1D1, n=33; C1D15, n=29; surgery, n=24) for 46 patients. The expression levels of 1,538 genes (493 up and 1,045 down) were significantly altered by anastrozole (Fig. 3A ). In contrast, only 6 genes, including KIF15, CASC5, FAM64A, TOP2A, ASPM and CEP55, were significantly altered (down-regulated) by adding palbociclib (C1D15 vs C1D1), which were also among the genes down-regulated by anastrozole and up-regulated at surgery (Fig 3B ). 235 (177 up and 58 down) genes were differentially expressed between surgery and C1D15. A number of classical ER regulated genes were down-regulated by anastrozole, including PDZK1, MAPT, PgR, STC2, RABEP1, TTF3, CCND1, PREX1, HSPB8, and RABEP1, which were not significantly altered by palbociclib (Supplementary Fig. S4 and Table S4 ). The top biological pathways altered by anastrozole (C1D1 vs. C0D1) included mitotic cell cycle, nuclear division, mitosis, and DNA replication, while the top pathways altered at the completion of neoadjuvant therapy (surgery vs. C1D15) also included programmed cell death and apoptotic process (Fig. 3C and Supplementary Table S4 ). Compared to C1D15, the surgical time point was associated with up-regulation of genes that promote cell cycle progression and a small set of genes that reduce apoptosis (Supplementary Table S4 ).

mRNA gene expression levels of G1 cyclins, CDKs and CDK inhibitors

We hypothesized that resistance to palbociclib was likely a result of deregulated G1/S cell cycle regulators. We therefore compared the mRNA expression levels of candidate genes, including RB1, CCND1, CCND2, CCND3, CCNE1, CDK2, CDK4, CDK6, CDKN2A, CDKN2B, CDKN2C, CDKN2D, CDKN1A and CDKN1B, by response groups and timepoints. Pairwise two-sample t-test analysis indicated significantly elevated expression of CCND3, CCNE1 and CDKN2D at C1D15 in the resistant group (Fig. 4 ). Interestingly, all three genes are transcriptionally regulated by E2F1 (30)(31)(32), suggesting persistent E2F activity in resistant tumors.

Discussion

NeoPalAna trial demonstrated the potent anti-proliferative effect of palbociclib in luminal breast cancers when the response to AI alone was incomplete. CCCA (Ki67≤2.7%) was achieved in 87% (90%CI 75-94%) at C1D15 (2 weeks post adding palbociclib), compared to the 28% (90%CI 17-41%) at C1D1 post 4 weeks of anastrozole monotherapy. The study met the primary endpoint in the overall population and in both PIK3CA WT (79%, 90%CI 62-90%), and Mut cohorts (100%, 90%CI 83-100%) (18,19). Significant improvement in the rates of CCCA and inhibition of cell proliferation were observed in both LumA and LumB subtypes, and regardless of PIK3CA mutation, menopausal status, tumor histology and grade. Palbociclib efficacy was observed across various genomic backgrounds that included somatic mutations in PTEN, TP53, and RUNX1 which have been associated with endocrine resistance (33). Notably the single HER2-E tumor which carried a RB1 p.E323fs frameshift mutation was resistant to palbociclib as expected from preclinical studies (12). Two tumors sensitive to palbociclib by Ki67 (>2.7% at C1D1, ≤2.7% at C1D15) were found to have missense RB1 mutations on baseline biopsies, PD131 (RB1 p.A562P and p.S576*) and PD203 (RB1 p.I532N -absent at subsequent time points). Although the sequencing depth were relatively high (92-400X), in the setting of low VAF, explanations for the disappearance of the RB1 p.I532N at subsequent time points include intra-tumoral heterogeneity in potentially spatially separated biopsies (34), clonal evolution in response to treatment (22), or sequencing artifact.

The broad anti-tumor activity of palbociclib in luminal breast cancers observed in this trial is consistent with findings in preclinical studies (12) and in clinical trials of patients with advanced breast cancer (13)(14)(15)(16). Although a previous presurgical window-of-opportunity study that randomized patients to receive 2 weeks of letrozole alone (Arm 1, n=2), or with ribociclib (Arm 2, 400mg daily, n=6), or ribociclib (Arm 3, 600mg daily, n=3) reported a mean decrease in Ki67 of 69% (38-100%), 96% (78-100%), and 92% (75-100%), in Arms 1, 2, and 3, respectively, the small sample size limited the ability to conclude on the added antiproliferative effect of ribociclib to letrozole (35). The current study therefore provides the initial biomarker evidence of enhanced anti-proliferative effect of a CDK4/6 inhibitor over that by an AI alone in primary breast cancers, supporting the investigation of palbociclib in the adjuvant setting for both pre-and postmenopausal women with luminal breast cancer.

ER+ breast cancer is enriched for activating mutations in PIK3CA (36,37) and there is significant interest in developing PI3K inhibitors based on promising preclinical data (38)(39)(40). However, the efficacy of pan-PI3K inhibitors in clinical trials has been limited by dose-limiting toxicities, including rash, diarrhea, and elevated transaminases (41), while alpha-specific inhibitors are still under clinical trial evaluation (NCT02340221 and NCT02437318). The potent anti-proliferative effect and well-tolerated safety profile of palbociclib in both PIK3CA mutant and WT populations observed in this trial is consistent with data from PALOMA-3 in which the addition of palbociclib to fulvestrant significantly improved PFS regardless of PIK3CA mutation status in patients with metastatic breast cancer by cell free tumor DNA analysis (42). Interestingly, all 6 resistant tumors in the current trial were PIK3CA WT. This apparent association, however, could be due to the fact that all PIK3CA mutant tumors analyzed were luminal, while 2 of the PIK3CA WT tumors were non-luminal. Since a large majority of PIK3CA WT tumors were responsive to palbociclib, the decision to use a CDK4/6 inhibitor should not be based on PIK3CA mutation status. However, it is possible that we might have enriched for a more resistant population in this trial due to the initial focus of enrollment to the PIK3CA WT cohort (43).

The rebound Ki67 at surgery was suppressed when palbociclib (cycle 5) was administered before surgery. This finding indicates that the anti-proliferative effect of palboclcib is reversible despite 4 months of therapy. Nevertheless, the data indicates continued dosing beyond 4 months will be needed in the adjuvant setting. Interestingly, one patient showed elevated Ki67 at surgery despite the initial CCCA at C1D15 and cycle 5 palbociclib, suggesting the development of acquired resistance.

To avoid pre-analytical, analytical, and scoring variations in Ki67 analysis, this trial employed a standardized sample acquisition method by providing biopsy/shipment kits, centralized processing, Ki67 staining, and pathologist-guided imaging analysis that have shown to yield reproducible Ki67 levels predictive of clinical outcomes (18). Importantly, the anti-proliferation effect of palbociclib over that of anastrozole alone based on Ki67 was replicated by the PAM50 11-gene proliferation score and the gene expression pathway analysis in this study, providing the feasibility and validity of centralized CLIA Ki67 as a biomarker endpoint in multi-center neoadjuvant trials.

Despite the eligibility requirement for ER rich tumors, the molecular characteristics of the tumor population were quite heterogeneous. The PAM50 analysis supports the use of palbociclib in both LumA and LumB breast cancers, however the benefit of palbociclib may be particularly important for LumB tumors as they more often exhibit persistent tumor proliferation on AI alone and carry a worse prognosis. Consistent with preclinical observations, neither the non-luminal breast cancers responded to palbociclib in this study (12). These data point to the potential value of PAM50 subtyping in clinical trials of CDK4/6 inhibitors.

The three response groups based on Ki67 levels at C1D1 and C1D15 clearly indicated that a subset of breast cancers (12/46, 26%) were able to achieve CCCA by anastrozole alone. However, no clear baseline biomarkers exist to date to identify this population for whom palbociclib could potentially be avoided (44,45). The sequential treatment approach and ontreatment biomarker analysis illustrated by NeoPalAna could therefore offer a platform for individualized response assessment and treatment recommendations.

In addition to non-luminal subtype as a potential resistant marker for palbociclib, gene expression analysis of G1 cyclins, CDKs and CDK inhibitors indicated that resistance to palbociclib appeared to be associated with persistently elevated on-treatment expression of CCND3, CCNE1 and CDKN2D. Interestingly, all three genes are known E2F1 transcription targets which are expected to be down-regulated upon CDK4/6 inhibition (30)(31)(32)46). The association between CCNE1 gain and resistance to CDK4/6 inhibition, likely through activation of CDK2, has been observed in preclinical studies (46). Our findings are hypothesis generating and warrant further investigations in larger sample sets.

Microarray analysis demonstrated that anastrozole significantly inhibited classical ER regulated genes and cell cycle pathways, findings consistent with previously reported neoadjuvant AI-induced gene expression changes (47)(48)(49). Although adding palbociclib further reduced cell proliferation, no significant change in the expression ER regulated genes was observed between C1D1 and C1D15, consistent with the selective action of palbociclib on CDK4/6. Strikingly, only 6 genes were further significantly reduced in their levels of expression at C1D15 compared to C1D1, illustrating the tight association between ER signaling and CDK4/6 activation.

This study has several limitations, including the moderate sample size and the lack of longterm follow up data to correlate with Ki67 response. Although we clearly observed three response categories with respect to sensitivity to anastrozole alone and to the addition of palbociclib, the small sample limited our ability to elucidate the underlying molecular mechanisms of palbociclib resistance. The findings of non-luminal subtype and persistent E2F target genes expression in palbociclib resistant tumors require further validation in other studies.

In summary, this 50-patient single arm, 2-cohort (PIK3CA WT and PIK3CA Mut), multicenter neoadjuvant phase II study provided proof of principle regarding the ability of CDK4/6 inhibition to overcome intrinsic endocrine resistance in primary breast cancer across a wide range of somatic mutation profiles. The association of treatment resistance with non-luminal subtype and persistent on-treatment expression of E2F targets including CCND3, CCNE1, and CDKN2D indicates persistent activation of E2F transcription in resistant tumors and warrants further investigation of alterative cell cycle inhibition approaches for this tumor subset.

Statement of Translational Relevance

Cyclin-dependent kinase (CDK) 4/6 plays an important role in driving cell cycle progression in estrogen receptor positive (ER+) breast cancer. The NeoPalAna trial therefore evaluated a CDK4/6 inhibitor in the neoadjuvant setting. Anastrozole monotherapy followed by the addition palbociclib allowed an individual assessment of the degree to which CDK4/6 inhibition added to aromatase inhibitor (AI) treatment. Additional proliferation suppression by palbociclib over anastrozole alone was observed across a wide range of clinicopathological and mutation backgrounds, including those with marked resistance to AI treatment. However palbociclib anti-proliferative effects were rapidly lost after CDK4/6 treatment was held for surgery thus justifying prolonged therapy in the adjuvant setting. The observation of palbociclib resistance in the two nonluminal ER+ breast cancers, including one with an RB1 deletion, indicates the importance of molecular subtype and RB1 status determination in patient selection.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgements

AcknowledgmentsThe authors thank the patients and families who participated in this study and the staff who cared for these patients.We thank Stephanie Myles for assistance in protocol development, the Siteman Cancer Center Tissue Procurement Core, McDonnell Genome Institute, Genomic and Pathology Service, and Anatomical and Molecular Pathology Laboratory.The study was funded by Pfizer Pharmaceutical, A Susan G. Komen Promise Grant (MJE), Siteman Cancer Center Grant (P30 CA91842, SCC, Eberlein), and Saint Louis Men's Group Against Cancer (Ma).Dr. Ma is a recipient of the NCI Clinical Investigator Team Leadership Award.Dr. OL Griffith was supported by the National Cancer Institute (NIH NCI K22CA188163).Dr. Ellis is a McNair Medical Foundation Scholar and the recipient of a Cancer Prevention Research Institute of Texas senior investigator award.This work is funded in part by Siteman Cancer Center Grant (P30 CA91842, SCC, Eberlein), NCI Cancer Clinical Investigator Team Leadership Award (3P30 CA091842-12S2, NIH/NCI, Ma), Susan G. Komen Promise Grant (Ellis), Saint Louis Men's Group Against Cancer (Ma), and Pfizer Pharmaceuticals.Dr. O. Griffith is supported by the National Cancer Institute (NIH NCI K22CA188163).Dr. Ellis is a McNair Medical Institute Scholar and a Cancer Prevention Institute of Texas Senior Investigator

Funding

Ma received research funding and advisory board member/consulting fees from Pfizer Pharmaceuticals and Novartis.M. Goetz received advisory/consulting fees for Eli Lilly. A. Forero received research funding from Pfizer Pharmaceuticals.M. Naughton served on Pfizer Pharmaceuticals speakers' bureaus.M. Ellis received advisory/consulting fees from Pfizer Pharmaceuticals and receives royalties from patents on the PAM50-based risk classifier (Prosigna).C. Huang and M. Koehler are employees of Pfizer Pharmaceuticals and have stock interest.

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