symposium article

Annals of Oncology 23 (Supplement 10): x219x227, 2012

doi:10.1093/annonc/mds307

Developments in Ki67 and other biomarkers

for treatment decision making in breast cancer
A. Sheri & M. Dowsett*
Department of Academic Biochemistry, Royal Marsden Hospital, London SW3 6JJ, UK

In recent decades, the widespread adoption of the use of

adjuvant therapy for breast cancer has led to a substantial
decline in breast cancer mortality [1]. Additionally, the
introduction of screening programs has led to the increasing
diagnosis of early-stage disease with an inherently better
prognosis. This challenges clinicians to identify patients
appropriately in whom adjuvant chemotherapy is warranted.
Optimal clinical decision making incorporates the use of
prognostic factors which identify risk independently of
treatment and also predictive makers which identify sensitivity
or resistance to a particular therapy. Several biomarkers such
as estrogen receptor, (ER), progesterone receptor (PgR) and
HER2 are established in breast cancer and routinely measured
at baseline. Their role and that of other less established
biomarkers are discussed in relation to treatment decision
making.
The administration of neoadjuvant therapy before surgery,
as well as facilitating breast conserving surgery, allows an
in vivo assessment of the primary tumours sensitivity to
systemic therapy. The absence of invasive tumour cells in the
excision specimen following neoadjuvant chemotherapy,
described as pathological complete response ( pCR), has been
validated as an intermediate marker of long-term outcome
with those patients whose tumours undergo a pCR having an
excellent long-term outcome [2, 3]. The neoadjuvant trial
design, where the pCR rate is often compared between
treatment arms, has been favoured as a method of testing the
*Correspondence to: Dr M. Dowsett. E-mail: mitch.dowsett@icr.ac.uk

activity of new agents in clinical development, requiring fewer

patients and less time than large-scale adjuvant studies.
However, failure to achieve a pCR following neoadjuvant
chemotherapy, particularly in ER-positive ( pos) cancers is
associated with a heterogeneous outcome. Furthermore, a pCR
in response to neoadjuvant endocrine therapy is uncommon
and extensive research has been undertaken to establish
biomarkers which predict long-term outcome based on the onor post-neoadjuvant therapy characteristics of residual disease.
This has the potential not only to improve prediction of longterm outcome but also to rationalise and accelerate the
development of new therapies for high-risk patients.

baseline biomarkers
estrogen and progesterone receptor
For over two decades, the expression of ER has been the
denitive biomarker for benet from endocrine therapy. With
the exception of the small minority of ER-negative (neg)
tumours positive for the PgR, ER-neg tumours do not derive
benet from endocrine therapy [46]. The most recent Early
Breast Cancer Trialists Collaborative Group (EBCTCG)
overview of trials evaluating 5 years of adjuvant tamoxifen
demonstrates a reduction in breast cancer mortality in ER-pos
disease of around a third throughout the rst 15 years [6].
Given the importance of ER and PgR expression on
treatment decision making and that reports have suggested up
to 20% of immunohistochemical ER and PgR testing
worldwide may be inaccurate, recent American Society of
Clinical Oncology/College of American Pathologists (ASCO/

The Author 2012. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
All rights reserved. For permissions, please email: journals.permissions@oup.com.

symposium
article

introduction

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Estrogen receptor (ER) and HER2 are well established as predictive markers for treatment benet, although
methodological deciencies can still affect their predictive accuracy. The shift towards earlier diagnosis poses a
challenge in identifying those low-risk patients who may safely avoid adjuvant chemotherapy for early breast cancer.
Therefore, recent research has focused on developing biomarkers to quantify residual risk on adjuvant endocrine
therapy. For widespread adoption into clinical practice, these must be validated in well-designed clinical trials and
provide additional information to current standards using reproducible and cost-effective methodologies. Furthermore,
evidence from preoperative studies indicates that on- or post-treatment biomarkers can be more predictive than at
baseline. In particular, Ki67 has recently emerged as an intermediate marker of long-term outcome. The power of Ki67
to predict treatment benet from endocrine therapy has facilitated the design of studies where Ki67 is the primary endpoint. This has also led to investigations into the predictive power of Ki67 to determine benet from signal transduction
inhibitors and chemotherapy in several recent and ongoing trials.
Key words: adjuvant therapy, biomarkers, breast cancer, IHC4, Ki67

symposium article

HER2
Amplication of the HER2 gene on chromosome 17q21 is
present in around 15% of breast cancers and before the clinical
use of trastuzumab was a strong predictor of poor outcome.
HER2 testing is now considered a standard as part of the
management of breast cancer. ASCO/CAP guidelines dene
HER2-amplied cases as those which demonstrate staining of
3+ by immunohistochemistry (IHC), a uorescent in situ
hybridisation (FISH) result of more than six HER2 gene copies
per nucleus or a FISH HER2:CEP17 ratio that is >2.2 [12]. An
equivocal result is dened as an IHC result of 2+, a FISH result
of 1.82.2 or 46 HER2 gene copies per nucleus. It should be
noted however, that patients with a HER2:CEP17 ratio of 2.0
were eligible for treatment with trastuzumab in the adjuvant

x | Sheri and Dowsett

trials of trastuzumab and so current evidence does not support

excluding these patients from trastuzumab therapy. A recent
study has reported that the degree of HER2 staining was not
correlated with long-term outcome following chemotherapy or
benet from trastuzumab [13].
HER2 amplication does appear to be associated with
chemotherapy sensitivity, with HER2 status reported to
independently predict pCR following neoadjuvant
chemotherapy [14]. Studies in the adjuvant setting have
suggested that the benet of anthracycline chemotherapy may
be conned to those patients with HER2-amplied tumours,
although a recent meta-analysis did not support this [15]. The
2011 St Gallen consensus guidelines do not recommend a
specic chemotherapy regime for HER2-amplied cancers
although the majority favoured the use of anthracyclines and
taxanes [16].
Whilst many HER2-positive cancers do appear
chemosensitive, recent evidence in the neoadjuvant setting
suggests that in some patients anti-HER2 therapy alone may be
sufcient. The NeoSphere study was a four-arm study testing
the addition of pertuzumab to trastuzumab, docetaxel or
trastuzumab and docetaxel compared with trastuzumab and
docetaxel. pCRs were observed in over 16% of tumours with
the combination of trastuzumab and pertuzumab alone [17].
Biomarkers to identify these patients are still lacking. Recent
analyses reported that higher HER2 membrane staining
determined using an H-score correlated with sensitivity to the
addition of pertuzumab to docetaxel and trastuzumab.
However the implications of this on the long-term outcome are
unknown [18].

baseline Ki67 and prognosis

A fundamental hallmark of cancer cells involves their ability to
sustain chronic proliferation [19, 20]. The most widely
practiced measurement of proliferation involves
immunohistochemical detection of the nuclear non-histone
protein Ki67. Its precise function remains ill-dened although
it is thought to be involved in ribosomal RNA synthesis [21,
22]. The observation that Ki67 is detected only in proliferating
cells and absent in quiescent cells led to its adoption as a
measure of the proportion of cells proliferating in a tumour.
Ki67 expression is commonly assessed using the mindbomb E3
ubiquitin protein ligase 1 antibody (MIB1) and reported as a
percentage of cells Ki67 positive.
Ki67 has been reported to correlate with other biomarkers in
breast cancer such as grade and ER expression, with ERpositive cancers typically exhibiting lower levels of proliferation
[23, 24]. Ki67 has also been used to identify luminal class with
a cut-off level of 13.25% proposed to distinguish poorer
prognosis luminal B cancers from luminal A [25]. However,
this cut-off only achieved a concordance of 75% with luminal
status and a lack of between laboratory standards may limit
application as a surrogate marker.
Numerous studies have investigated the potential role of
Ki67 as a prognostic marker. In a meta-analysis of 40 studies
involving over 11 000 patients, baseline Ki67 was found to have
a modest prognostic value in multivariable analysis, which was
more evident in lymph node-negative patients [26]. Another

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CAP) guidelines focus on standardised testing of ER and PgR

in breast cancer [7]. It is recommended that ER and PgR assays
may be considered positive if there are at least 1% positive
tumour nuclei in the presence of expected reactivity in
controls. Furthermore, recommendations are made for external
quality assurance procedures requiring laboratories to undergo
prociency testing.
Quantitative expression of ER has been investigated as a
predictive marker of endocrine therapy benet. Studies in
advanced disease from the 1970s have correlated a greater
benet from endocrine therapy with higher concentrations of
ER [8]. In the adjuvant setting, the most recent EBCTCG
meta-analysis reported no apparent benet from tamoxifen for
tumours classied as ER poor (<10 fmol/mg), but a substantial
benet in marginally ER-pos disease (1019 fmol/mg),
measured using a ligand-binding assay. Highly ER-pos disease
(200 fmol/mg) was associated with an even greater benet
with a rate ratio for breast cancer mortality with tamoxifen of
0.53 compared with 0.67 in marginally ER-pos disease [6].
These results are also consistent with the observation that
higher mRNA expression of ESR1 correlates with greater
benet from tamoxifen in the NSABP B-14 trial [9].
The expression of ER has also been investigated as a marker
of benet from chemotherapy. Lower rates of pCR are
observed in ER-pos cancers following neoadjuvant
chemotherapy and some studies have reported a smaller
proportional benet from adjuvant chemotherapy in ER-pos
compared with ER-neg tumours. However, this is not borne
out in meta-analyses and the recent EBCTCG overview
reported a similar proportional benet for adjuvant
chemotherapy in ER-pos and ER-neg tumours. The potential
benet from chemotherapy in ER-pos disease therefore
depends on the absolute risk without chemotherapy on
appropriate endocrine therapy.
It is important to note that ER-pos, PgR-neg cancers are
associated with a worse prognosis. However, analyses suggest
that they derive a similar proportional benet from adjuvant
tamoxifen or aromatase inhibitors compared with ER-pos,
PgR-pos tumours [6, 10, 11]. Therefore, although PgR
expression does not predict proportional benet from
endocrine therapy, it may be useful in determining residual
risk and therefore may be an aid in adjuvant chemotherapy
treatment decisions.

Annals of Oncology

Annals of Oncology

meta-analysis of 46 studies including over 12 000 patients

found that Ki67 positivity (using cut-offs dened by individual
authors) was associated with a higher risk of relapse and a
worse survival in patients with early breast cancer [27].
Standardised methodologies for measurement and cut-off
points for Ki67 are lacking which has limited the evaluation
and application of this biomarker in clinical practice. As a
result, the ASCO Tumour Marker Guidelines Committee
determined that evidence supporting the clinical utility of Ki67
was insufcient to recommend routine use for prognostic
purposes in patients with newly diagnosed breast cancer [28].
In 2011, the International Ki67 in Breast Cancer Working
Group published recommendations for Ki67 assessment in
breast cancer [29]. These guidelines aim to minimise preanalytical and analytical variables in Ki67 assessment and
harmonise scoring methodology and data handling.

Other studies have focused on the role of Ki67 in predicting

response to adjuvant therapy. Specic questions investigated
have been whether baseline Ki67 predicts benet from
adjuvant chemotherapy or can even predict benet from a
specic agent.
Despite studies suggesting that high Ki67 is associated with a
poor prognosis, high Ki67 has been associated with a good
response to neoadjuvant chemotherapy. Although in
multivariable analyses, not all studies have shown Ki67 to be
an independent predictor of pCR [30]. Furthermore, Ki67
alone has not been shown to predict the benet of adjuvant
cyclophosphamide, methotrexate and uorouracil (CMF)
chemotherapy to adjuvant endocrine therapy in lymph nodenegative patients [31]. Other studies have examined the power
of baseline Ki67 to predict benet from specic regimes in
patients treated with adjuvant chemotherapy reporting a trend
towards benet from the addition of taxanes to anthracyclinebased chemotherapy in Ki67 high versus Ki67 low cancers
which warrants further investigation [32, 33].
Studies of neoadjuvant endocrine therapy have not reported
a clear association with baseline Ki67 and response to therapy
[34]. However, a higher Ki67 has been associated with a
shorter time to treatment failure in patients with advanced
breast cancer treated with an aromatase inhibitor [35].
Additionally in a central analysis of the BIG1-98 trial, a higher
Ki67 was associated with a trend towards a greater
proportional benet from letrozole versus tamoxifen [36].

identifying residual risk in ER-pos breast cancer

treated with endocrine therapy and prediction of
chemotherapy benet
With the advent of screening, a large number of women are
currently diagnosed with small ER-pos breast cancers, many of
which have a generally good prognosis. Accurate determination
of residual risk in patients treated with endocrine therapy
allows low-risk patients to safely be spared the toxicity of
chemotherapy. Several multigene signatures have been
developed which have been shown to be prognostic and predict
chemotherapy benet. However, these are costly and may not

Volume 23 | Supplement 10 | September 2012

necessarily inform adjuvant therapy decisions more than more

established markers. It is recognised that ER, PgR, HER2 and
Ki67 measured using immunohistochemistry independently
provide some prognostic and sometimes predictive
information. The IHC4 incorporates these established markers
along with nodal status, tumour size, grade and age into a
prognostic score for ER-pos breast cancer patients receiving
endocrine treatment. In an analysis of the arimidex, tamoxifen,
alone or in combination (ATAC) trial and subsequently
validated in a separate cohort, the IHC4 was highly prognostic.
Furthermore, the prognostic information provided by the
IHC4 was similar to that provided by the 21-gene recurrence
score (RS), which is weighted to measure ER, HER2 and
proliferation-associated genes [37]. Similarly, Mammostrat is a
tool measuring ve immunohistochemical markers but unlike
with the IHC4, these are not routinely measured in clinical
practice (P53, HTF9C, NDRG1, and SLCA5 and CEACAM5).
Mammostrat scores have been shown to be prognostic in ERpos breast cancer and predict the degree of benet in the
NSABP B20 trial [38, 39]. No comparison has been made with
the RS. As with the RS, an intermediate group is identied in
whom the degree of benet is uncertain. Incorporation of
standard clinico-pathological variables such as tumour size and
nodal involvement may allow more accurate determination of
prognosis and therefore absolute benet from chemotherapy.

on and post-treatment biomarkers

Ki67 and preoperative endocrine therapy
Ki67 has been studied extensively as a biomarker to predict
long-term outcome and to assess potential therapeutic efcacy
in the neoadjuvant setting (see Table 1 for the summary of
endocrine studies) [4051]. On- or post-treatment
measurements of Ki67 in residual disease appear to integrate
information on both intrinsic tumour biology and
responsiveness to therapy and are more predictive of long-term
outcome following neoadjuvant endocrine therapy than pretreatment measures.
In the IMPACT trial, higher expression of Ki67 after just 2
weeks of endocrine therapy was found to be associated with a
statistically signicant lower recurrence-free survival (RFS)
where as higher Ki67 at baseline was not [42]. The ongoing
POETIC trial is prospectively testing the hypothesis that Ki67
measured after 2 weeks of endocrine therapy can predict longterm outcome. Similarly, post-treatment Ki67 has been
incorporated into the preoperative endocrine prognostic index
(PEPI) score along with ER expression and the post-treatment
measures of residual disease burden, pathological T and N
stage [52]. Moreover, differences in Ki67 suppression during
preoperative endocrine therapy have been shown to predict
differential treatment effects on long-term outcome in the
adjuvant setting. The greater suppression of Ki67 with letrozole
versus tamoxifen in the PO24 study and anastrozole over the
combination with tamoxifen or tamoxifen alone in IMPACT
mirrored the results of the BIG 1-98 and ATAC trials,
respectively [53, 54]. More recently, the American College of
Surgeons Oncology Group Z1031 study demonstrated the
equivalence of the aromatase inhibitors exemestane,

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baseline Ki67 and prediction of adjuvant therapy

benet

symposium article

symposium article

Annals of Oncology

Table 1. Preoperative studies of endocrine agents assessing change in Ki67 as an endpoint.

Reference

Study Design

Biomarker endpoints

Results
No signicant differences in OR.
Greater suppression of Ki67 at 2 and
12 weeks with anastrozole compared
to tamoxifen (p= 0.004 and p=
0.001).
No difference between tamoxifen
and combination.
Higher Ki67 at 2 weeks associated
with lower RFS p= 0.004
Greater OR rate in letrozole compared
to tamoxifen group p < 0.001.
Greater reduction in Ki67 with
letrozole compared to tamoxifen p
= 0.0009.
No differences in Ki67 suppression or
PEPI score.

Smith et al.
[40]
Dowsett
et al. 42,
43]

Double blind. randomised, phase 3 . 12 weeks

anastrozole 1mg, tamoxifen20mg or
combination od in non metastatic ER-pos
postmenopausal breast cancer.

Objective
response (OR)
rate at 12
weeks

Reduction in Ki67 at 2
and 12 weeks. Ki67
expression at 2 weeks.

Eiermann
et al. [44]
Ellis et al.
[45]

Double blind randomised phase 2b/. 16 weeks

letrozole 2.5 mg or tamoxifen 20mg od in
postmenopausal ER-pos locally advanced breast
cancer.

OR at 16 weeks

Reduction in Ki67 at 16
weeks

Ellis et al.
[46]

Randomised phase2.
Exemestane 25mg, letrozole 2.5mg or
anastrozole 1 mg od in postmenopausal ER-pos
stage II-III breast cancer.
Double blind, randomized phase 3. Goserelin +
anastrozole 1mg or tamoxifen 20 mg od in
premenopausal primary breast cancer.

Clinical response Reduction in Ki67 at 16at 16-18

18 weeks.
weeks.
PEPI score.

Open label, randomized phase2 . Fulvestrant

500mg (day 0, 14, 28 every 28 days) vs 250mg (
day 0, 28 every 28 days) in postmenopausal ERpos locally advanced breast cancer.
Double blind, randomised. Idoxifene 40mg od vs
placebo in postmenopausal primary breast
cancer.

Change in Ki67
from baseline
to week 4.

Ki67
ER and PgR
expression.

Change in Ki67
at 14-21 days.

Double blind, randomized, placebo controlled

phase 2.
Postmenopausal stage I or II primary breast
cancer. Raloxifene 60mg od, 300mg bd or
placebo.

Change in Ki67
at 14 days.

Ki67.
Change in apoptotic
index.
ER and PgR
expression.
Ki67
Apoptosis
ER and PgR
expression

Masuda et al.
[47]

Kuter et al.
[48]

Dowsett et al.
[49]

Dowsett et al.
[50]

Clinical response Reduction in Ki67 at 24

at 24 weeks.
weeks.

anastrozole and letrozole on Ki67 suppression mirroring

disease-free survival (DFS) results of the adjuvant MA.27 trial
[46, 55].
An exception to this trend is the study of Tibolone effects on
Mammary carcinoma tissue (STEM) trial, where the
proliferative effects of tibolone were compared against placebo.
No signicant differences were observed at 14 days in Ki67
expression [51]. These results were not consistent with the
increase in breast cancer recurrence observed in ER-pos early
breast cancer patients in the Livial Intervention following
breast cancer: efcacy, recurrence and tolerability end-points
(LIBERATE) study. This may relate to the time point assessed
(14 days) which may in some cases be too early to identify the
proliferative effects of a hormone replacement therapy or the
early acquired escape from the anti-proliferative effects of

x | Sheri and Dowsett

Higher rate of clinical response in

anastrozole vs tamoxifen groups p=
0.004.
Greater reduction in Ki67 with
anstrozole vs tamoxifen p < 0.0001.
Greater reduction in Ki67 with 500mg
vs 250mg fulvestrant at 16 weels p =
<0.0001.
Reduction in KI67 in ER-pos tumours
p = 0.0043.
No effect in ER-neg or placebo
groups.
No signicant effect on apoptosis.
In ER-pos tumours: Reduction in Ki67
with raloxifene 60mg vs placebo p=
0.015.
Reduction in Ki67 raloxifene 600mg
vs placebo p= 0.064.
No signicant effect on apoptosis.

endocrine therapy. In the IMPACT study, 15% of patients

with suppression of Ki67 at 2 weeks demonstrated a rebound
effect with higher levels measured at 12 weeks. Therefore,
although failure to suppress Ki67 at 2 weeks is likely to identify
patients with an adverse prognosis, suppression at 2 weeks may
not necessarily always indicate a good prognosis. This recovery
in some patients by 12 weeks may, however, be exploited in
assessing the ability of new agents to prevent this Ki67
recovery.

Ki67 and signal transduction inhibitors

The predictive power of Ki67 in the neoadjuvant endocrine
setting has led to the extension of its use as a potential
biomarker for the efcacy of several signal transduction

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Primary
Endpoint

symposium article

Annals of Oncology
Table 2. Preoperative studies of signalling agents assessing change in Ki67 as an endpoint.
Reference
Smith et al.
[56]

Guix et al.
[57]

DeCensi et al.
[60]

Martin and
Davies
et al. [61]
Macaskill
et al. [62]

Baselga et al.
[63]

Biomarker
endpoints

Clinical
endpoints

Change in Ki67
OR rate
Postmenopausal patients with stage I-IIb ER or
at 2 and 16
PgR -pos breast cancer randomized to
weeks.
anastrozole + getinib 250mg od for 16 weeks,
placebo for 2 weeks followed by getinib for 14
weeks or placebo for 16 weeks.
n/a
Preoperative window of opportunity study. Primary Change in Ki67
breast cancer patients with stage I-IIIa primary
Apoptosis
breast cancer treated with erlotinib 150mg od 6P-EGFR, P10 days.
HER2
P-MAPK, PAKT
PS6
S118 P-ER
Clinical
Change in Ki67.
HER2 + breast cancers > 4cm.
response
Apoptosis
NCT00133796: Neoadjuvant trastuzumab 4mg/
P-AKT
m2 then weekly at 2mg/m2 for 3 weeks then
P-MAPK
addition of docetaxel100mg/m2 every 3 weeks
PTEN, PIK3CA
for 4 cycles.
mutation
NCT00206427: Neoadjuvant lapatinib 1500mg
od for 6 weeks followed by docetaxel 100mg/m2
every 3 weeks for 4 cycles.

Preoperative window of opportunity study In

HER2 + breast cancer.
Lapatinib 1500mg od vs placebo for 3 weeks.

Primary
endpoint:
Change in
Ki67.
ER, PgR,
HER2, PTEN
Phase 2 randomised placebo controlled window of Change in Ki67
opportunity study. Postmenopausal patients with
Apoptosis
stage I-II primary breast cancer. Celecoxib
ER, PgR,
400mg bd.
COX-2, CD31.
Preoperative window of opportunity study in
Change in Ki67
postmenopausal patients with breast cancer.
Apoptosis
5mg Everolimus od for 14 days.
P-AKT
P-MTOR
P-S6
HER2
Phase II neoadjuvant study of everolimus 10mg od/ Change in Ki67
placebo plus letrozole 2.5mg od.
PIK3CA
mutation
P-S6
Cyclin D1
PR

inhibitors in neoadjuvant and short-term pre-surgical window

of opportunity studies (see Table 2) [5663]. However, the
ability of Ki67 to predict benet from agents whose main
mechanism of action may not necessarily always be directly
anti-proliferative is less certain.

Volume 23 | Supplement 10 | September 2012

Radiological
response

Results
No clinical or biological effect of the addition of
neoadjuvant getinib to anastrozole.

Signicant inhibition of Ki67 in ER-pos tumours

only p= 0.0006.
Reduction in P-MAPK, P-AKT, PS6 and S118
P-ER in ER + tumours.

Lapatinib alone: Reduction in Ki67 with

associated reduction in P-MAPK. No
alteration in apoptosis index. Higher rate of
pCR associated with low PTEN p= 0.007. No
association with PIK3CA mutation.
Trastuzumab : Signicant increase in
apoptosis index at week 1 p = 0.004. No
alteration in Ki67. Non signicant trend
towards lower incidence of pCR in low PTEN
tumours. Non signicant trend towards lower
incidence of pCR with PIK3CA mutation.
Signicant decrease in Ki67 in lapatinib arm vs
placebo . Signicant increase in Ki67 observed
in placebo arm p= 0.008.

n/a

Non-statistically signicant trend toward

reduction in Ki67. No alteration in apoptosis.

Toxicity.

Signicant decrease in Ki67 observed

p= 0.019.
High baseline Ki67, HER2 positivity and
reduction in P-AKT associated with reduction
in Ki67.

Clinical
response.

Greater mean reduction in Ki67 with everolimus

+ letrozole vs letrozole alone p = 0.0002.

Ki67 and other biomarkers to predict long-term

outcome following neo-adjuvant chemotherapy
post-treatment Ki67

High Ki67 in residual disease following neoadjuvant

chemotherapy correlates with poor long-term outcome

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Mohsin et al.
[58]
Dave et al.
[59]

Study Design

symposium article

Annals of Oncology

Table 3. Neo-adjuvant chemotherapy studies assessing change in Ki67 as an endpoint.

Reference

Study Design

Biomarker endpoints

Clinical endpoints

Retrospective analysis of single centre cohort of Change in Ki67

patients treated with neoadjuvant
Pre and Post Ki67
chemotherapy.
ER, PgR, HER2

RFS
OS

Colleoni
et al. [67]

Retrospective anlalysis of single centre cohort

of patients treated with neoadjuvant
chemotherapy.

pCR
ER, PgR, HER2,
Ki67

Billgren
et al. [68]

Single centre study of patients treated with 3-4

cycles of cyclophosphamide, epirubicin and
5-uoruracil chemotherapy.

Ki67 pre and before

2nd course of
chemotherapy.

DFS
Clinical and
radiological
response.
OR rate.

Bottini et al.
[69]

Retrospective single institution study of

patients treated with neoadjvuant
chemotherapy in phase 2 studies of CMF +
concurrent tamoxifen in ER-pos disease or
single agent epirubicin.
Patients treated with neoadjuvant uorouracil,
epirubicin, cyclophosphamide (FEC)
chemotherapy.

Change in and Ki67

pre and post.
ER and PgR
HER2, BCL2.

RFS

Pre, post and change

in Ki67.
Apoptosis
pCR

OR

Burcombe
et al. [70]

[6466]. One of the largest series published is from the Royal

Marsden where in a cohort of 284 patients, post-therapy Ki67
was highly prognostic, with those patients with an excision
Ki67 in the highest tertile, having a 5-year relapse-free survival
of just 27% and overall survival (OS) of 39% compared with
77% and 93%, respectively, in the lowest Ki67 tertile. In an
analysis of 103 matched pre- and post-treatment samples,
reduction in Ki67 from pre-treatment to excision was also
correlated with long-term outcome although less so than the
excision reading.
Other studies have reported partially conicting results
regarding the prognostic signicance of change in Ki67 with
neoadjuvant chemotherapy (see Table 3) [64, 6770]. Time of
measurement after a course of chemotherapy may be a critical
variable in interpreting these results. Whilst, differences in pCR
rates are commonly used to assess the efcacy of chemotherapy
or targeted therapy combinations in the neoadjuvant setting,
no studies have yet reported that Ki67 measured in residual
disease or change in Ki67 following neoadjuvant chemotherapy
can predict the differential treatment effects of agents on longterm outcome.
on-treatment markers

Fewer studies have examined the role of on- rather than posttreatment biomarkers of response to neoadjuvant
chemotherapy. Clinical response following two cycles of
chemotherapy has not been shown to reliably predict
pathological response [71]. Clinical studies also indicate that
apoptosis can be detected within 2448 h of commencing
chemotherapy with some studies correlating these changes

x | Sheri and Dowsett

Results
Excision Ki67 signicantly associated with
RFS and OS in multivariate analysis.
Change in Ki67 (combined prognostic
index) correlated with RFS and OS.
No correlation between modication of Ki67
and clinical response.

Decrease of more than 25% in Ki67 after 1st

course of chemotherapy correlated with
decreased risk of disease recurrence p =
0.033. Decrease in Ki67 after 1st course
added prognostic information in
multivariable analysis.
Ki67 at surgery rather than baseline better
predictor of RFS.
Reduction in Ki67 not correlated with RFS.

No association between clinical and

pathological response and change in Ki67
at 21 days.

with the pathological response. However, these changes have

not been correlated with long-term outcome [7274]. A
marker of homologous recombination competence, RAD51,
measured 24 h after the rst dose of chemotherapy has also
been investigated as a marker of chemotherapy sensitivity, with
33% of cancers with low RAD51 scores achieving a pCR in one
neoadjuvant study [75]. Additionally, a small 33 patient study
from the Karolinska institute reported that a change in Ki67 of
>25% after just one cycle of cyclophosphamide, epirubicin and
5-uoruracil chemotherapy signicantly correlated with a
decreased risk of disease recurrence, P = 0.033 [76].

RCB and tumour cellularity

In 2003, Miller and Payne published a ve-point grading
system based on the changes in tumour cellularity in the
excised tumour compared with the diagnostic core biopsy to
predict long-term outcome following neoadjuvant
chemotherapy. To account for potential heterogeneity in
tumour cellularity, at least three pre-treatment core biopsies
were taken from different areas of the tumour. Grade of
response was signicantly correlated with DFS (P = 0.02) and
OS (P = 0.01) [77]. The RCB extended this concept of
measuring tumour cellularity in residual post-chemotherapy
disease, incorporating the dimensions of residual tumour bed
and nodal involvement into a prognostic index. This score
classies patients into one of three risk categories, with patients
with minimal residual disease (class I) having the same
prognosis as those with a pCR [78]. A pathological response
index (PRI) which includes the presence of vascular invasion

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Jones et al.
[64]

Annals of Oncology

and evidence of chemotherapy-related changes along with

reduction in the tumour size and the presence of positive
apical lymph node metastases was able to differentiate RCB
class II patients into good and poor prognostic sub-groups
[79].
Meaningful reporting of residual disease following
neoadjuvant therapy, particularly in ER-pos breast cancers, is
likely to not only improve prognostication in these patients,
but also will potentially further inform interpretation of
neoadjuvant trials. Furthermore, if high-risk patients are
clearly identied on the basis of post-neoadjuvant therapy
characteristics, this would also facilitate the design of postneoadjuvant adjuvant studies tailored to the molecular prole
of high-risk residual disease.

circulating markers

conclusions
Recent years have seen advances in the molecular
understanding of the biology of breast cancer with the
development of several prognostic and predictive multigene
signatures. Before clinical application, these require appropriate
validation against the current standards and demonstration of
reproducible methodologies. The established biomarkers such
as ER and HER2 remain powerful predictors of response to
therapy. Whilst in many instances insufcient to be used in
isolation, the integration of several markers using wellvalidated methodologies may allow more accurate risk
estimation and therefore aid adjuvant chemotherapy decision
making.
The development of on- or post-treatment biomarkers of
response has the potential to improve prognostication even
further. Moreover, if intermediate markers such as Ki67 were
shown to have general applicability in predicting the long-term
outcome, the impact on trial design could be substantial.
Current adjuvant studies require large numbers of patients and
often many years before results become apparent. Reliable
prediction of drug efcacy in the preoperative setting therefore
has the potential to accelerate the development of novel
treatment strategies.

Volume 23 | Supplement 10 | September 2012

acknowledgements
We are grateful for funding from the Royal Marsden NIHR
Biomedical Research Centre.

disclosures
AS has no conict of interest that could be perceived as
prejudicing the impartiality of the research reported. MD has
acted as an advisor for Astrazeneca, Roche, GSK, Sano and
Ipsen.

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