Abstract
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 anti-proliferative 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 1mg daily for 4 weeks (cycle 0) (with goserelin if premenopausal), followed by adding palbociclib (125mg 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 inadequately response. Anastrozole was continued until surgery, which occurred 3-5 weeks post palbociclib exposure. Later patients received additional 10-12 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 clinicopathological and mutation profiles. Ki67 recovery at surgery following palbociclib washout was suppressed by cycle 5 palbociclib. Resistance was associated with non-luminal subtypes and persistent E2F-target gene expression.
Conclusions
Palbociclib is an active anti-proliferative agent for early-stage breast cancer resistant to anastrozole, however, prolonged administration may be necessary to maintain its effect.
Keywords: palbociclib, anastrozole, neoadjuvant, breast cancer, CDK4/6
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) (1-4). 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 (6-9). 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 anti-tumor 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 (13-16). 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.
Patient Population and Methods
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 HIV-positivity, 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 (2nd 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 (3rd 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 (21-23). 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 time-points (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.
Results
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).
Table 1. Patient and Tumor Characteristics.
| Entire Cohort (n=50) | PIK3CA Mut (n=16) | PIK3CA WT (n=32) | |
|---|---|---|---|
|
| |||
| Median Age (range) years | 57.5 (34.1-79.6) | 55.2 (34.1-79.3) | 57.7 (34.8-79.6) |
|
| |||
| Race | |||
| White | 47 (94%) | 16 (100%) | 29 (91%) |
| Black | 3 (6%) | 0 | 3 (9%) |
|
| |||
| Menopausal Status | |||
| Premenopausal | 18 (36%) | 8 (50%) | 11 (34%) |
| Postmenopausal | 32 (64%) | 8 (50%) | 21 (66%) |
|
| |||
| PgR Allred Score | |||
| Neg | 3 (6%) | 0 | 3 (9%) |
| Pos | 47 (94%) | 16 (100%) | 29 (91%) |
|
| |||
| Tumor grade | |||
| 1 | 12 (24%) | 4 (25%) | 7 (22%) |
| 2 | 31 (62%) | 11 (69%) | 19 (59%) |
| 3 | 7 (14%) | 1 (6%) | 6 (19%) |
|
| |||
| Histology | |||
| Ductal | 33 (66%) | 11 (69%) | 21 (66%) |
| Lobular/mixed | 15 (30%) | 4 (25%) | 10 (31%) |
| Other (papillary, mucinous) | 2 (4%) | 1 (6%) | 1 (3%) |
|
| |||
| Clinical Stage | |||
| II/IIA | 20 (40%) | 6 (38%) | 14 (44%) |
| IIB | 16 (32%) | 8 (50%) | 7 (22%) |
| IIIA | 12 (24%) | 2 (12%) | 9 (28%) |
| IIIB/C | 2 (4%) | 0 | 2 (6%) |
|
| |||
| PAM50 Intrinsic Subtype | N=32 | N=9 | N=23 |
| LumA | 17 (53%) | 5 (56%) | 12 (52%) |
| LumB | 12 (38%) | 3 (33%) | 9 (39%) |
| HER2-E | 1 (3%) | 0 | 1 (4%) |
| Basal-like | 1 (3%) | 0 | 1 (4%) |
| Normal | 1 (3%) | 1 (11%) | 0 |
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 dose-level 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).
Table 2. Rate Of Ccca At C1D1 And C1D15, Overall And By Key Subgroups.
| Variable | Levels | CCCA at C1D1 | CCCA at C1D15 | P value | CCCA at C1D15 in anastrozole resistant casesa | |||
|---|---|---|---|---|---|---|---|---|
| N/total | % (90% CI) | N/total | % (90% CI) | N/total | % (90% CI) | |||
| All evaluable patients | 12/46 | 26% (16-39%) | 39/45 | 87% (75-94%) | <0.001 | 26/31 | 84% (69-93%) | |
| PIK3CA | WT | 7/28 | 25% (12-42%) | 22/28 | 79% (62-90%) | <0.001 | 14/19 | 74% (52-89%) |
| Mutant | 4/16 | 25% (9-48%) | 16/16 | 100% (83-100%) | <0.001 | 12/12 | 100% (78-100%) | |
| QNS | 1/2 | 50% (NE) | 1/1 | 100% (NE) | -- | -- | -- | |
| Baseline Intrinsic Subtype | LumA | 6/15 | 40% (19-64%) | 16/16 | 100% (83-100%) | 0.008 | 9/9 | 100% (72-100%) |
| LumB | 1/11 | 9% (0-36%) | 9/12 | 75% (47-93%) | 0.020 | 8/10 | 80% (49-96%) | |
| Normal | 1/1 | 100% (NE) | 1/1 | 100% (NE) | -- | -- | ||
| Non Luminal | 0/2 | 0% (NE) | 0/2 | 0% (NE) | -- | 0/2 | 0% (NE) | |
| Menopausal status | Pre- | 6/18 | 33% (16 -55%) | 15/17 | 88% (67 - 98%) | 0.003 | 9/11 | 82% (53-97%) |
| Post- | 6/28 | 21% (10 -38%) | 24/28 | 86% (70 - 95%) | <0.001 | 17/20 | 85% (66-96%) | |
| Histology | Ductal | 3/30 | 10% (3-24%) | 25/30 | 83% (68-93%) | <0.001 | 20/25 | 80% (62-92%) |
| Lobular/Mixed | 8/14 | 57% (33-79%) | 12/13 | 92% (68-100%) | 0.103 | 5/5 | 100% (55-100%) | |
| Others | 1/2 | 50% (NE) | 2/2 | 100% (NE) | -- | 1/1 | 100% (NE) | |
| Tumor Grade | G1 | 4/11 | 36% (14-65%) | 10/11 | 91% (64-100%) | 0.025 | 5/6 | 83% (42-100%) |
| G2 | 7/28 | 25% (12-42%) | 24/27 | 89% (74-97%) | <0.001 | 17/19 | 89% (70-98%) | |
| G3 | 1/7 | 14% (0-52%) | 5/7 | 71% (34-95%) | 0.046 | 4/6 | 67% (27-94%) | |
| PgR | Positive | 12/43 | 28% (17-41%) | 38/42 | 90% (80-97%) | <0.001 | 25/28 | 89% (75-97%) |
| Negative | 0/3 | 0% (NE) | 1/3 | 33% (NE) | -- | 1/3 | 33% (NE) | |
| PTEN | WT | 9/31 | 29% (16-45%) | 27/31 | 87% (73-95%) | <0.001 | 18/21 | 86% (67-96%) |
| Mutant | 1/8 | 13% (0-47%) | 7/8 | 88% (53-100%) | 0.014 | 6/7 | 86% (48-100%) | |
| TP53 | WT | 8/30 | 27% (14-43%) | 28/30 | 93% (80-99%) | <0.001 | 20/21 | 95% (79-100%) |
| Mutant | 2/9 | 22% (4-55%) | 6/9 | 67% (34-90%) | 0.046 | 4/7 | 57% (23-87%) | |
| RB1 | WT | 10/36 | 28% (16-43%) | 32/36 | 89% (76-96%) | <0.001 | 22/25 | 88% (72-97%) |
| Mutant | 0/3 | 0% (NE) | 2/3b | 67% (NE) | -- | 2/3b | 67% (NE) | |
Anastrozole resistance was defined as Ki67>2.7% (non-CCCA) at C1D1.
Two (PD203 and PD131) of the 3 RB1 mutant tumors had low frequency RB1 mutations and were resistant to anastrozole (C1D1 Ki67>2.7%) but sensitive to palbociclib (Ki67<2.7%). PD203 (Ki67: baseline 36%, C1D1 27%, C1D15 34%) had RB1 p.532N with VAFs (depth of coverage) of 5.4% (92×) at baseline, which was not identified at subsequent time points (C1D1, C1D15 and surgery). PD131 (Ki67: baseline 2.7%, C1D1 22%, C1D15 1%) had RB1 p.A562P (VAF 4.7%) and p.S576* (VAF 6.8%) at baseline (coverage 400×). Sequencing data for subsequent time points were not available for PD131. PD106 was resistant to both anastrozole and palbociclib (Ki67: baseline 38%, C1D1 40%, C1D15 48%) had RB1 p.E323fs with VAF of 11% (baseline) and 25% (C1D15) (∼30×). NE, not evaluable.
Fig 1. Ki67 response by PIK3CA mutation status (A-C), luminal subtype (D-F) and with/without Cycle 5 (G-I).
Box plots of all evaluable samples of indicated cohort (A, D, G) and Ki67 of individual tumors (B-C, E-F, H-I) are shown. *p<0.005 comparing Ki67 values with that of the previous time-point in the corresponding cohort. Line colors in the Ki67 graphs denote response category (Anastrozole Sensitive, C1D1 Ki67<2.7%; Palbociclib Sensitive, C1D1 Ki67≤2.7% and C1D15 Ki67<2.7%; Resistant, C1D15 Ki67≤2.7%).
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 co-occurring 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. Somatic mutation, intrinsic subtype and clinicopathological characteristics of individual tumors in relation to Ki67 response.
Genes with mutations detected in at least 2 samples are shown. Mutation frequency, number of tier 1 mutations.
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 11-gene 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).
Fig 3. Microarray gene expression analysis.
A, Venn-diagram of the number of genes significantly changed (Benjamini-Hochberg false discovery rate adjusted F test P≤0.05) between time-points. B, Heatmap of the 6 genes significantly down-regulated by anastrozole and by adding palbociclib. C, Top 20 significantly altered Go pathways comparing the gene expression profiles between C1D1 and C0D1, or between surgery and C1D15. % indicates the percentage of genes observed in the indicated GO pathway.
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 time-points. 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 (30-32), suggesting persistent E2F activity in resistant tumors.
Fig 4. Boxplots of gene expression levels of CCND3, CCNE1, and CDKN2D by Ki67 response and by time-point.
Significant p-values by pairwise two sample t-test were indicated. The sample sizes for anastrozole sensitive, palbociclib sensitive, and resistant groups were 6, 18, and 5 at C0D1, 6, 21, and 3 at C1D1, 5, 20, and 3 at C1D15, and 3, 17, 1 at surgery time-points, respectively.
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 (13-16). 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 anti-proliferative 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 (38-40). 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 on-treatment 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 (30-32, 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 (47-49). 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 long-term 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), multi-center 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.
Supplementary Material
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 non-luminal ER+ breast cancers, including one with an RB1 deletion, indicates the importance of molecular subtype and RB1 status determination in patient selection.
Acknowledgments
The 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
Footnotes
Presented in part at the 2015 Annual San Antonio Breast Cancer Symposium
Conflict of Interest Statement: C. 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. All other authors disclosed no potential conflict of interest.
References
- 1.Sherr CJ. Cancer cell cycles. Science. 1996;274:1672–7. doi: 10.1126/science.274.5293.1672. [DOI] [PubMed] [Google Scholar]
- 2.Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999;13:1501–12. doi: 10.1101/gad.13.12.1501. [DOI] [PubMed] [Google Scholar]
- 3.van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science. 1993;262:2050–4. doi: 10.1126/science.8266103. [DOI] [PubMed] [Google Scholar]
- 4.Weinberg R. The retinoblastoma protein and cell cycle control. Cell. 1995;81:323–30. doi: 10.1016/0092-8674(95)90385-2. [DOI] [PubMed] [Google Scholar]
- 5.Weintraub SJ, Prater CA, Dean DC. Retinoblastoma protein switches the E2F site from positive to negative element. Nature. 1992;358:259–61. doi: 10.1038/358259a0. [DOI] [PubMed] [Google Scholar]
- 6.Altucci L, Addeo R, Cicatiello L, Germano D, Pacilio C, Battista T, et al. Estrogen induces early and timed activation of cyclin-dependent kinases 4, 5, and 6 and increases cyclin messenger ribonucleic acid expression in rat uterus. Endocrinology. 1997;138:978–84. doi: 10.1210/endo.138.3.5002. [DOI] [PubMed] [Google Scholar]
- 7.Geum D, Sun W, Paik SK, Lee CC, Kim K. Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: differential induction mechanism of cyclin D1 and D3. Molecular Reprod. Dev. 1997;46:450–8. doi: 10.1002/(SICI)1098-2795(199704)46:4<450::AID-MRD2>3.0.CO;2-N. [DOI] [PubMed] [Google Scholar]
- 8.Said TK, Conneely OM, Medina D, O'Malley BW, Lydon JP. Progesterone, in addition to estrogen, induces cyclin D1 expression in the murine mammary epithelial cell, in vivo. Endocrinology. 1997;138:3933–9. doi: 10.1210/endo.138.9.5436. [DOI] [PubMed] [Google Scholar]
- 9.Tong W, Pollard JW. Progesterone inhibits estrogen-induced cyclin D1 and cdk4 nuclear translocation, cyclin E- and cyclin A-cdk2 kinase activation, and cell proliferation in uterine epithelial cells in mice. Mol Cell Biol. 1999;19:2251–64. doi: 10.1128/mcb.19.3.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Thangavel C, Dean JL, Ertel A, Knudsen KE, Aldaz CM, Witkiewicz AK, et al. Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer. Endocr Relat Cancer. 2011;18:333–45. doi: 10.1530/ERC-10-0262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade M, Trachet E, et al. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther. 2004;3:1427–38. [PubMed] [Google Scholar]
- 12.Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, et al. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast Cancer Res. 2009;11:R77. doi: 10.1186/bcr2419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Finn RS, Martin M, Rugo HS, Jones S, Im SA, Gelmon K, et al. Palbociclib and Letrozole in Advanced Breast Cancer. N Engl J Med. 2016;375:1925–36. doi: 10.1056/NEJMoa1607303. [DOI] [PubMed] [Google Scholar]
- 14.Finn RS, Crown JP, Ettl J, Schmidt M, Bondarenko IM, Lang I, et al. Efficacy and safety of palbociclib in combination with letrozole as first-line treatment of ER-positive, HER2-negative, advanced breast cancer: expanded analyses of subgroups from the randomized pivotal trial PALOMA-1/TRIO-18. Breast Cancer Res. 2016;18:67. doi: 10.1186/s13058-016-0721-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Turner NC, Ro J, Andre F, Loi S, Verma S, Iwata H, et al. Palbociclib in Hormone-Receptor-Positive Advanced Breast Cancer. N Engl J Med. 2015 doi: 10.1056/NEJMoa1505270. [DOI] [PubMed] [Google Scholar]
- 16.Finn RS, Crown JP, Lang I, Boer K, Bondarenko IM, Kulyk SO, et al. The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol. 2015;16:25–35. doi: 10.1016/S1470-2045(14)71159-3. [DOI] [PubMed] [Google Scholar]
- 17.Ellis MJ, Tao Y, Luo J, A'Hern R, Evans DB, Bhatnagar AS, et al. Outcome prediction for estrogen receptor-positive breast cancer based on postneoadjuvant endocrine therapy tumor characteristics. J Natl Cancer Inst. 2008;100:1380–8. doi: 10.1093/jnci/djn309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Ellis MJ, Suman VJ, Hoog J, Goncalves R, Sanati S, Creighton CJ, et al. J Clin Oncol. 2017. Ki67 roliferation Index as a Tool for Chemotherapy Decisions During and After Neoadjuvant Aromatase Inhibitor Treatment of Breast Cancer: Results From the American College of Surgeons Oncology Group Z1031 Trial (Alliance) p. JCO2016694406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Ellis MJ, Suman VJ, Hoog J, Lin L, Snider J, Prat A, et al. Randomized phase II neoadjuvant comparison between letrozole, anastrozole, and exemestane for postmenopausal women with estrogen receptor-rich stage 2 to 3 breast cancer: clinical and biomarker outcomes and predictive value of the baseline PAM50-based intrinsic subtype--ACOSOG Z1031. J Clin Oncol. 2011;29:2342–9. doi: 10.1200/JCO.2010.31.6950. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Nielsen TO, Parker JS, Leung S, Voduc D, Ebbert M, Vickery T, et al. A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor-positive breast cancer. Clin Cancer Res. 2010;16:5222–32. doi: 10.1158/1078-0432.CCR-10-1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Ma CX, Luo J, Naughton M, Ademuyiwa F, Suresh R, Griffith M, et al. A Phase I Trial of BKM120 (Buparlisib) in Combination with Fulvestrant in Postmenopausal Women with Estrogen Receptor–Positive Metastatic Breast Cancer. Clin Cancer Res. 2016;22:1583–91. doi: 10.1158/1078-0432.CCR-15-1745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Miller CA, Gindin Y, Lu C, Griffith OL, Griffith M, Shen D, et al. Aromatase inhibition remodels the clonal architecture of estrogen-receptor-positive breast cancers. Nat Commun. 2016;7:12498. doi: 10.1038/ncomms12498. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Griffith M, Griffith OL, Smith SM, Ramu A, Callaway MB, Brummett AM, et al. Genome Modeling System: A Knowledge Management Platform for Genomics. PLoS Comput Biol. 2015;11:e1004274. doi: 10.1371/journal.pcbi.1004274. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Skidmore ZL, Wagner AH, Lesurf R, Campbell KM, Kunisaki J, Griffith OL, et al. GenVisR: Genomic Visualizations in R. Bioinformatics. 2016;32:3012–4. doi: 10.1093/bioinformatics/btw325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Liang KY, Zeger SL. Longitudinal data analysis using generalized linear models. Biometrika. 1986;73:13–22. [Google Scholar]
- 26.Hochberg Y, Tamhane AC. Multiple Comparison Procedures. John Wiley & Sons, Inc; 2008. [Google Scholar]
- 27.Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47. doi: 10.1093/nar/gkv007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Falcon S, Gentleman R. Using GOstats to test gene lists for GO term association. Bioinformatics. 2007;23:257–8. doi: 10.1093/bioinformatics/btl567. [DOI] [PubMed] [Google Scholar]
- 29.Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 2010;12:R68. doi: 10.1186/bcr2635. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Ma Y, Yuan J, Huang M, Jove R, Cress WD. Regulation of the Cyclin D3 Promoter by E2F1. J Biol Chem. 2003;278:16770–6. doi: 10.1074/jbc.M212702200. [DOI] [PubMed] [Google Scholar]
- 31.Geng Y, Eaton EN, Picon M, Roberts JM, Lundberg AS, Gifford A, et al. Regulation of cyclin E transcription by E2Fs and retinoblastoma protein. Oncogene. 1996;12:1173–80. [PubMed] [Google Scholar]
- 32.Carcagno AL, Marazita MC, Ogara MF, Ceruti JM, Sonzogni SV, Scassa ME, et al. E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation. PloS one. 2011;6:e21938. doi: 10.1371/journal.pone.0021938. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Ellis MJ, Ding L, Shen D, Luo J, Suman VJ, Wallis JW, et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature. 2012;486:353–60. doi: 10.1038/nature11143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Gellert P, Segal CV, Gao Q, Lopez-Knowles E, Martin LA, Dodson A, et al. Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation. Nat Commun. 2016;7:13294. doi: 10.1038/ncomms13294. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Curigliano G, Gómez Pardo P, Meric-Bernstam F, Conte P, Lolkema MP, Beck JT, et al. Ribociclib plus letrozole in early breast cancer: A presurgical, window-of-opportunity study. The Breast. 2016;28:191–8. doi: 10.1016/j.breast.2016.06.008. [DOI] [PubMed] [Google Scholar]
- 36.Saal LH, Holm K, Maurer M, Memeo L, Su T, Wang X, et al. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res. 2005;65:2554–9. doi: 10.1158/0008-5472-CAN-04-3913. [DOI] [PubMed] [Google Scholar]
- 37.Network TCGA. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61–70. doi: 10.1038/nature11412. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Crowder RJ, Phommaly C, Tao Y, Hoog J, Luo J, Perou CM, et al. PIK3CA and PIK3CB inhibition produce synthetic lethality when combined with estrogen deprivation in estrogen receptor-positive breast cancer. Cancer Res. 2009;69:3955–62. doi: 10.1158/0008-5472.CAN-08-4450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Miller TW, Balko JM, Fox EM, Ghazoui Z, Dunbier A, Anderson H, et al. ERalpha-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer. Cancer Discov. 2011;1:338–51. doi: 10.1158/2159-8290.CD-11-0101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Ma CX, Crowder RJ, Ellis MJ. Importance of PI3-kinase pathway in response/resistance to aromatase inhibitors. Steroids. 2011;76:750–2. doi: 10.1016/j.steroids.2011.02.023. [DOI] [PubMed] [Google Scholar]
- 41.Krop IE, Mayer IA, Ganju V, Dickler M, Johnston S, Morales S, et al. Pictilisib for oestrogen receptor-positive, aromatase inhibitor-resistant, advanced or metastatic breast cancer (FERGI): a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Oncol. 17:811–21. doi: 10.1016/S1470-2045(16)00106-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Cristofanilli M, Turner NC, Bondarenko I, Ro J, Im SA, Masuda N, et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol. 2016;17:425–39. doi: 10.1016/S1470-2045(15)00613-0. [DOI] [PubMed] [Google Scholar]
- 43.Vora SR, Juric D, Kim N, Mino-Kenudson M, Huynh T, Costa C, et al. CDK 4/6 inhibitors sensitize PIK3CA mutant breast cancer to PI3K inhibitors. Cancer Cell. 2014;26:136–49. doi: 10.1016/j.ccr.2014.05.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Ma CX, Reinert T, Chmielewska I, Ellis MJ. Mechanisms of aromatase inhibitor resistance. Nat Rev Cancer. 2015;15:261–75. doi: 10.1038/nrc3920. [DOI] [PubMed] [Google Scholar]
- 45.Ma CX, Bose R, Ellis MJ. Prognostic and Predictive Biomarkers of Endocrine Responsiveness for Estrogen Receptor Positive Breast Cancer. Adv Exp Med Biol. 2016;882:125–54. doi: 10.1007/978-3-319-22909-6_5. [DOI] [PubMed] [Google Scholar]
- 46.Herrera-Abreu MT, Palafox M, Asghar U, Rivas MA, Cutts RJ, Garcia-Murillas I, et al. Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer. Cancer Res. 2016;76:2301–13. doi: 10.1158/0008-5472.CAN-15-0728. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47.Miller WR, Larionov A, Anderson TJ, Evans DB, Dixon JM. Sequential changes in gene expression profiles in breast cancers during treatment with the aromatase inhibitor, letrozole. Pharmacogenomics J. 2012;12:10–21. doi: 10.1038/tpj.2010.67. [DOI] [PubMed] [Google Scholar]
- 48.Miller WR, Larionov AA, Renshaw L, Anderson TJ, White S, Murray J, et al. Changes in breast cancer transcriptional profiles after treatment with the aromatase inhibitor, letrozole. Pharmacogenet Genomics. 2007;17:813–26. doi: 10.1097/FPC.0b013e32820b853a. [DOI] [PubMed] [Google Scholar]
- 49.Mackay A, Urruticoechea A, Dixon JM, Dexter T, Fenwick K, Ashworth A, et al. Molecular response to aromatase inhibitor treatment in primary breast cancer. Breast Cancer Res. 2007;9:R37. doi: 10.1186/bcr1732. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.