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UC San Francisco Previously Published Works
Title
Ki67 Proliferation 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).
Permalink
https://escholarship.org/uc/item/5qb5216t
Journal
Journal of clinical oncology : official journal of the American Society of Clinical
Oncology, 35(10)
ISSN
0732-183X
Authors
Ellis, Matthew J
Suman, Vera J
Hoog, Jeremy
et al.
Publication Date
2017-04-01
DOI
10.1200/jco.2016.69.4406
Peer reviewed
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University of California
JOURNAL OF CLINICAL ONCOLOGY
ORIGINAL REPORT
Ki67 Proliferation 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)
Matthew J. Ellis, Vera J. Suman, Jeremy Hoog, Rodrigo Goncalves, Souzan Sanati, Chad J. Creighton, Katherine
DeSchryver, Erika Crouch, Amy Brink, Mark Watson, Jingqin Luo, Yu Tao, Michael Barnes, Mitchell Dowsett,
G. Thomas Budd, Eric Winer, Paula Silverman, Laura Esserman, Lisa Carey, Cynthia X. Ma, Gary Unzeitig,
Timothy Pluard, Pat Whitworth, Gildy Babiera, J. Michael Guenther, Zoneddy Dayao, David Ota, Marilyn Leitch,
John A. Olson Jr, D. Craig Allred, and Kelly Hunt
ABSTRACT
Purpose
To determine the pathologic complete response (pCR) rate in estrogen receptor (ER) –positive primary
breast cancer triaged to chemotherapy when the protein encoded by the MKI67 gene (Ki67) level
was . 10% after 2 to 4 weeks of neoadjuvant aromatase inhibitor (AI) therapy. A second objective was
to examine risk of relapse using the Ki67-based Preoperative Endocrine Prognostic Index (PEPI).
Methods
The American College of Surgeons Oncology Group (ACOSOG) Z1031A trial enrolled post-
menopausal women with stage II or III ER-positive (Allred score, 6 to 8) breast cancer whose
treatment was randomly assigned to neoadjuvant AI therapy with anastrozole, exemestane, or
letrozole. For the trial ACOSOG Z1031B, the protocol was amended to include a tumor Ki67 de-
termination after 2 to 4 weeks of AI. If the Ki67 was . 10%, patients were switched to neoadjuvant
chemotherapy. A pCR rate of . 20% was the predefined efficacy threshold. In patients who
completed neoadjuvant AI, stratified Cox modeling was used to assess whether time to recurrence
differed by PEPI = 0 score (T1 or T2, N0, Ki67 , 2.7%, ER Allred . 2) versus PEPI . 0 disease.
Results
Only two of the 35 patients in ACOSOG Z1031B who were switched to neoadjuvant chemotherapy
experienced a pCR (5.7%; 95% CI, 0.7% to 19.1%). After 5.5 years of median follow-up, four (3.7%)
of the 109 patients with a PEPI = 0 score relapsed versus 49 (14.4%) of 341 of patients with PEPI . 0
(recurrence hazard ratio [PEPI = 0 v PEPI . 0], 0.27; P = .014; 95% CI, 0.092 to 0.764).
Conclusion
Chemotherapy efficacy was lower than expected in ER-positive tumors exhibiting AI-resistant
proliferation. The optimal therapy for these patients should be further investigated. For patients with
PEPI = 0 disease, the relapse risk over 5 years was only 3.6% without chemotherapy, supporting the
study of adjuvant endocrine monotherapy in this group. These Ki67 and PEPI triage approaches are
being definitively studied in the ALTERNATE trial (Alternate Approaches for Clinical Stage II or III
Estrogen Receptor Positive Breast Cancer Neoadjuvant Treatment in Postmenopausal Women: A
Phase III Study; clinical trial information: NCT01953588).
J Clin Oncol 35:10 61-1069. © 2017 by American Society of Clinical Oncology. Creative Commons
Attribution Non-Commercial No Derivati ves 4.0 License:
https://creativecomm ons.org /licenses/by-
nc-nd/4.0/
INTRODUCTION
For postmenopausal women with clinical stage II
or III estrogen receptor (ER) –positive breast
cancer, neoadjuvant aromatase inhibition (AI) is
an underused and low-toxicity alternative to
chemotherapy for increasing breast conservation
rates.
1
A barrier to greater adoption of neo-
adjuvant AI is high response var iability. We
therefore postulated that an early sw itch from
AI to neoadjuvant chemotherapy could produce
Author affiliations and support information
(if applicable) appear at the end of this
article.
Published at
jco.org on January 3, 2017.
M.J.E. and V.J.S. are joint first authors.
The content is solely the responsibility of
the authors and does not necessarily
represent the official views of the National
Institutes of Health or the companies
involved.
Clinical trial information: NCT00265759.
Corresponding author: Matthew J. Ellis,
MB, BChir, PhD, Lester and Sue Smith
Breast Center, One Baylor Plaza,
MSBCM600, Houston, TX 77030; e-mail:
mjellis@bcm.edu.
© 2017 by American Society of Clinical
Oncology. Creative Commons Attribution
Non-Commercial No Derivatives 4.0
License.
0732-183X/17/3510w-1061w/$20.00
ASSOCIATED CONTENT
See accompanying Editorial
on page
1031
Appendix
DOI: 10.1200/JCO.2016.69.4406
Data Supplements
DOI: 10.1200/JCO.2016.69.4406
DOI: 10.1200/JCO.2016.69.4406
© 2017 by American Society of Clinical Oncology 1061
VOLUME 35
•
NUMBER 10
•
APRIL 1, 2017
better clinical outcomes for tumors that responded poorly to AI.
Conversely, adjuvant AI alone may be sufficient to prevent relapse
in tumors highly responsive to neoadjuvant AI.
Pathologic complete response (pCR) after systemic chemo-
therapy remains a controversial clinical trial end point,
2,3
and al-
ternatives are needed, particularly in ER-positive, human epidermal
growth factor receptor 2–negative disease (HER2-negative), where
pCR rates are low. Data from a neoadjuvant study comparing
letrozole and tamoxifen in postmenopausal women with ER-positive
breast cancer—P024 (Preoperative Treatment of Postmenopausal
Breast Cancer Patients with Letrozole: A Randomized Double-
Blind Multicenter Study)
4-6
—was previously used to generate the
Preoperative Endocrine Prognostic Index (PEPI).
7
PEPI requires
pathologic stage (tumor size and nodal status), in addition to levels
of the protein encoded by the MKI67 gene (Ki67), and Allred ER
score measured on the surgical specimen (with surgery conducted
during uninterrupted endocrine therapy). Patients with a PEPI score
of 0 (pT1 or pT2, pN0, Ki67 # 2.7%, Allred score . 2) from the
P024 trial were found to have a low risk of relapse. Similar findings
were observed in the neoadjuvant IMPACT trial (Immediate Pre-
operative Anastrozole, Tamoxifen, or Combined with Tamoxifen)
2
,
but no subsequent validation efforts have been reported.
The American College of Surgeons Oncology Group (ACO-
SOG) Z1031A randomized phase II clinical trial was designed to
determine which AI (anastrozole, letrozole, or exemestane) should
be recommended for future testing against chemotherapy in the
neoadjuvant setting (ACOSOG is now a part of the Alliance for
Clinical Trials in Oncology). The major initial finding from
ACOSOG Z1031A was that half of the patients who were con-
sidered candidates for mastectomy or inoperable before neo-
adjuvant AI therapy had successful breast-conserving surgery.
8
When the enrollment in ACOSOG Z1031A was complete, an
amendment was introduced (ACOSOG Z1031B) that triaged
patients with tumors exhibiting a Ki67 . 10% in a tumor biopsy 2
to 4 weeks after starting AI to standard chemotherapy. The hy-
pothesis being tested was that the pCR rate would be at least 20% in
this AI-resistant population. Herein, we report the pCR results
from ACOSOG Z1031B as well as the time to recurrence by PEPI
status among all ACOSOG Z1031 patients who completed neo-
adjuvant AI treatment.
METHODS
Establishment of an Early Ki67 Cut Point for Triage to
Chemotherapy
An on-treatment Ki67 threshold for switching from neoadjuvant AI
therapy to neoadjuvant chemotherapy was established using data from the
Preoperative Letrozole study (POL)
9
and the IMPACT trial.
10
A Ki67 value
of . 10% at 1 month in the POL study was associated with a higher PEPI
score (P = .01), a smaller number of patients in the PEPI-0 group (P = .08),
and worse relapse-free survival (P = .0016). Similarly, the IMPACT data
confirmed that a 2-week Ki67 . 10% predicted a higher PEPI score
(P = .001), smaller numbers of patients in the PEPI-0 group (P = .004), and
worse relapse-free survival (P = .008; Data Supplement; Appendix
Table A1
and Figure A1A, online only). Combining these studies revealed only one
PEPI-0 case among 51 patients with a 2- to 4-week Ki67 value of . 10%.
Thus, according to the PEPI model, patients with a Ki67 value of 10% at 2
to 4 weeks had a , 2% chance of a favorable PEPI score that would allow
them to safely avoid chemotherapy under current guidelines.
Patient Eligibility
Eligible patients were postmenopausal with invasive breast cancer
(clinical stage T2 to T4c, N0 to N3, M0). Additional criteria have been
previously described.
8
This study was supported by the Clinical Trials
Support Unit and approved by the institutional review boards of all
participating institutions. Each participant signed an institutional review
board–approved, protocol-specific informed consent in accordance with
federal and institutional guidelines.
Treatment Schema
Patients enrolled in ACOSOG Z1031B underwent a core breast bi-
opsy for Ki67 determination after 2 weeks of AI therapy. If the Ki67 was
# 10%, the patient continued AI therapy for another 12 to 14 weeks and
then proceeded to surgery. Women whose 2-week Ki67 level was . 10%
were offered either a National Comprehensive Cancer Network–approved
neoadjuvant chemotherapy regimen or surgery at the discretion of their
providers and/or the patients. If the biopsy core contained insufficient
tumor to perform the Ki67 assay, patients could elect to undergo a repeat
biopsy at 4 weeks or continue on AI therapy. If severe treatment-related
toxicity was reported or the patient refused further AI therapy, surgery
was recommended. Within 14 days of registration, patients underwent
a complete physical examination with tumor assessment. Every 4 weeks,
patients underwent a physical examination, toxicity assessment, and tumor
assessment using February 2000 WHO criteria. If tumor progression was
suspected, ultrasound or mammogram was required, and neoadjuvant AI
was discontinued if progression was confirmed. Blood and biopsy spec-
imens for correlative studies were collected at baseline, 2 to 4 weeks after
the start of neoadjuvant AI treatment, on discontinuation of neoadjuvant
treatment, and at surgery. For patients with PEPI = 0 disease, management
without chemotherapy was recommended but not mandatory to de-
termine the acceptability of this recommendation.
AI Treatment
Before the release of the ACOSOG Z1031A results,
8
eligible patients
were randomly assigned to 16 to 18 weeks of neoadjuvant AI with
exemestane 25 mg once daily, letrozole 2.5 mg once daily, or anastrozole
1 mg once daily. After the release of the data, patients could choose either
letrozole or anastrozole treatment.
Statistical Considerations
The primary end point for ACOSOG Z1031B was the pCR rate
among the women who after 2 weeks of neoadjuvant AI therapy had
a tumor Ki67 level of . 10% and switched to neoadjuvant chemotherapy.
pCR was defined as histolog ic evidence of no invasive tumor cells in the
surgical breast specimen or ipsilateral lymph nodes. A one-stage phase II
clinical trial desig n was chosen to assess the pCR rate. With a sample size of
35 and a one-sided a = 0.10, a one-sample binomial test of proportions
would have a 90% chance of declaring success with a pCR rate of at least
20% compared with the null hypothesis that the pCR was 5% (at least four
pCRs were needed to conclude pCR rate $ 20%). A 90% binomial
confidence interval for the true pCR was also constructed. On the basis of
the IMPACTstudy, it was estimated that 235 eligible women would need to
enroll to obtain 35 women with a 2-week Ki67 . 10% willing to switch to
neoadjuvant chemotherapy. The long-term PEPI outcome study cohort
excluded women from both ACOSOG Z1031A and Z1031B who withdrew
consent not having started neoadjuvant AI, had metastases or bilateral
invasive breast cancer at registration, failed to undergo surgery, had al-
ternative therapy before surgery, had radiographic confirmed disease
progression or new primary disease during neoadjuvant AI, failed to have
sentinel lymph node or axillary lymph node dissection, or lacked sufficient
tissue to obtain a Ki67 or Allred score. In addition, ACOSOG Z1031B
patients with 2- or 4-week Ki67 . 10% who remained on AI were ex-
cluded. Time to breast cancer recurrence (TBCR) was defined as the time
from surgery to first local, regional, or distant disease recurrence. Patients
1062 © 2017 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY
Ellis et al
without documented disease recurrence were censored at the date of their
last disease evaluation. TBCR was estimated using the Kaplan-Meier
method
11
Stratified Cox modeling (with cohort and adjuvant chemo-
therapy use as strata) was used to assess whether TBCR differed w ith
respect to P EPI 0 status.
12
The Alliance Statistics and Data Center
conducted data collection and statistical analyses. Data were locked
on January 11, 2016.
Ki-67 CLIA Assay for ACOSOG Z1031A and Z1031B
For both ACOSOG Z1031 cohorts, a Ki67 clinical trial assay was
performed at the College of American Pathologists– and the Clinical
Laboratory Improvement Amendments–certified Washington University
anatomic and molecular pathology laborator y using the CONFIRM anti-
Ki67 (30-9) rabbit monoclonal primary antibody as a prediluted reagent
on a Benchmark XT platform according to the manufacturer instructions
(Ventana, Tucson, AZ). Tonsil was used as the assay positive control.
Ki-67 Quantification Approaches
A single experienced pathologist (D.C.A.) conducted the real-time
Ki67 scoring for ACOSOG Z1031B. If the estimated rate was low (, 2.7%),
or high (. 10%), a whole slide estimate was conducted. If the score was
between 2.7% and 10%, point counting was conducted using an ocular
grid, at least three high-power fields with a minimum of 100 cells scored.
Retrospective analysis of ACOSOG Z1031A used the iScan Coreo scanner
(Ventana) with the Companion Algorithm Ki-67 (30-9) software. The
imaging approach required three to 10 areas of interest be selected at 43
magnification excluding ductal carcinoma in situ, vessels, and lymphocytes
and avoiding perinecrotic or necrotic areas. The image analysis result was
reviewed to ensure the software was correctly differentiating between benign
and malignant cells, and, if not, the case was triaged to visual point counting.
The visual point counting approach required color photomicrographs with
a background grid taken at 403 of at least three fields selected based on
invasive tumor content and the quality of the histology, not on Ki67 staining
pattern, to obtain tumor cell count of at least 200. The scorer counted the
total number of tumor cells and the number of Ki67-positive cells that
intersected with first grid line and every third gridline thereafter.
Gene Expression Analysis to Study Cell Cycle–Regulated
Genes
RNA preparation and Agilent 44K gene array analysis(Agilent
Technologies, Santa Clara, CA) approaches were carried out as previously
described.
2
The microarray contained probes for 720 of the 874 genes
previously identified as having periodic expression in the cell division cycle
of HeLa cells (Data Supplement).
13
Gene expression levels in each tumor
were normalized to the number of standard deviations from the median
expression value across all the tumors. A multigene proliferation score
Table 1. Patient and Disease Characteristics for the ACOSOG Z1031B Cohort Sorted by Early on-Treatment Ki67 Categories
Characteristic Week-2 Ki67 # 10% (n = 165) Week-2 Ki67 . 10% (n = 49)
Week-2 Biopsy, No Invasive
Disease Present (n = 22)
Median age, years (IQR) 65 (58-72) 60 (55-64) 66 (58-72)
Histology
Ductal 114 (69.1) 40 (81.6) 16 (72.7)
Lobular 34 (20.6) 7 (14.3) 4 (18.2)
Mixed ductal/lobular 10 (6.1) 2 (4.1) 0
Other 7 (4.2) 0 2 (9.1)
Grade
1 54 (32.7) 13 (26.5) 8 (36.4)
2 96 (58.2) 22 (44.9) 12 (54.5)
3 15 (9.1) 14 (28.6) 2 (9.1)
HER2 positive 3 (1.8) 2 (4.1) 1 (4.5)
cTstage
T2 131 (79.4) 32 (65.3) 19 (86.4)
T3 27 (16.4) 16 (32.6) 2 (9.1)
T4A-C 7 (4.2) 1 (2.1) 1 (4.5)
cNstage
N0 113 (68.5) 27 (55.1) 21 (95.5)
N1 46 (27.9) 19 (38.8) 1 (4.5)
N2 4 (2.4) 3 (6.1) 0
N3 1 (0.6) 0 0
Unknown 1 (0.6) 0 0
Neoadjuvant endocrine therapy
Anastrozole 72 (43.6) 16 (32.7) 2 (9.1)
Exemestane 21 (12.7) 10 (20.4) 10 (45.4)
Letrozole 72 (43.6) 23 (46.9) 10 (45.4)
Week 2 Ki67 value
0-2.5 89 (53.9) 0
2.6-5.0 44 (26.7) 0
5.1-7.5 20 (12.1) 0
7.6-10.0 12 (7.3) 0
10.1-15.0 0 19 (38.8)
15.1-20.0 0 12 (24.5)
20.1-25.0 0 6 (12.2)
25.1-50.0 0 8 (16.3)
50.1-75.0 0 1 (2.0)
75.1-100 0 3 (6.1)
NOTE. Data presented as No. (%) unless otherwise noted.
Abbreviations: HER2, human epidermal growth factor receptor 2; IQR, interquartile range; Ki67, protein encoded by the MKI67 gene.
jco.org © 2017 by American Society of Clinical Oncology 1063
Ki67-Based Decisions for Breast Cancer Neoadjuvant Endocrine Therapy
(MGPS) for a tumor was the average normalized expression of the 772
genes for that tumor analysis.
14
The microarray data sets have been
submitted to Gene Expression Omnibus (GSE87411).
RESULTS
ACOSOG Z1 031B P atient Cohort
From October 1, 2009 to November 15, 2011, 245 patients were
enrolled into ACOSOG Z1031B. At week 2, 165 women (69.9%) had
aKi67valueof# 10%; 49 women (20.8%) had a Ki67 value . 10%,
and22women(9.3%)hadinsufficient tumor to make a Ki67 de-
termination. Pa tient and disease characteristics of these 236 women
are presented in
Table 1 and the CONSOR T diagram in Figure 1.
Neoadjuvant Chemotherapy Efficacy for ER-Positive
Tumors Exhibiting a 2- to 4-Week Ki67 Value > 10%
After Sta rting Aromatase Inhibition
Among the 49 patients whose week 2–Ki67 was . 10%, 35
patients switched to neoadjuvant chemotherapy; three patients
continued with AI, eight patients went directly to surgery, two
patients went to surgery after a rebiopsy at week 4 and again found
their Ki67 to be . 10%; one patient pursued treatment outside of
this study. Twenty-five (71.4%) of the 35 had an anthracycline-
containing regimen (
Table 2). Six patients (17.1%) failed to
complete their planned course of chemotherapy because of in-
tolerability. There were two (5.7%; 95% CI, 0.7% to 19.1%) pCRs
among these 35 patients. A pCR occurred in a 55-year-old woman
with cT2N0 ductal breast cancer with a 2-week Ki67 of 80.0%
treated with doxorubicin and cyclophosphamide (A C) plus docetaxel.
The second pCR was in a 59-year-old woman with cT3N1 ductal
breast cancer with a 2-week Ki67 of 31.7% subsequently treated with
docetax el plus gemcitabine plus bevacizumab followed by A C plus
bevacizumab.
Neoadjuvant Outcomes for ACOSOG Z1031B Patients
WhoseWeek2to4Ki67Was£ 10%
There were 187 patients whose 2-week Ki67 value was
either # 10% (165) or not determined because of insufficient
tumor in the biopsy specimen (22). One of 187 patients whose
week 2–Ki67 value was # 10% chose to go directly to surgery
rather than continue on AI. Of 22 patients whose 2-week Ki67
value was not obtained, all chose to remain on AI either after
a rebiopsy at week 4 Ki67 of # 10% (six patients) or after forgoing
a rebiopsy at 4 weeks (16 patients; see
Fig 1 for exact disposition
of all patients). Among the remaining 177 patients, pathologic
evaluation revealed no residual disease in the breast or lymph
nodes in three patients, in only the breast in 92 patients, and in
both the breast and lymph nodes in 82 patients (
Table 3). The pCR
rate among the 186 women who completed AI was therefore 1.6%
(95% CI, 0.3% to 4.6%). PEPI scores were: 0 in 64 patients; 1 to 7 in
109 patients; and not determined in 13 patients because of pro-
gression during neoadjuvant AI therapy (two patients), lack of Allred
score or Ki67 from surgical specimen (four patients), or failure to
undergo surgery for reasons other than progression (seven patients).
Thus, the PEPI-0 rate was 34.4% (95% CI, 27.6% to 41.7%).
Registration
(N = 245)
Breast biopsy after 2 weeks of NET
(n = 236)
Unable to ascertain Ki67 as
no invasive tumor in
biopsy specimen
(n = 22)
Ki67 ≤ 10%
(n = 165)
Ki67 > 10%
(n = 49)
Continue AI,
no week-4
biopsy
(n = 16)
Continue AI,
week-4 Ki67
≤ 10% so
continued AI
(n = 6)
Continue AI
(n = 164)
Surgery
(n = 1)
Chose to
continue AI
(n = 3)
Continued
AI, week-4
Ki67 also
>10%, chose
surgery
(n = 2)
Surgery
(n = 8)
Off study
(n = 1)
Switched to
NAC
(n = 35)
Cancelled participation before start of treatment
Ineligible because receiving treatment for cervical cancer
Bilateral disease
(n = 3)
(n = 1)
(n = 2)
Withdrew consent before week-2 biopsy
Discontinued because of intolerability before week-2 biopsy
(n = 2)
(n = 1)
Fig 1. CONSORT diagram for the ACOSOG Z1031B patients. AI, aromatase inhibitor; Ki67, protein encoded by the MKI67 gene; NAC, neoadjuvant chemotherapy; NET,
neoadjuvant endocrine therapy.
1064 © 2017 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY
Ellis et al