Neoadjuvant chemotherapy with or without zoledronic acid ......Mar 19, 2013 · Neoadjuvant chemotherapy + zoledronic acid in breast cancer 6 Materials and Methods Study Patients

Neoadjuvant chemotherapy + zoledronic acid in breast cancer

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Neoadjuvant chemotherapy with or without zoledronic acid in early breast

cancer – a randomised biomarker pilot study.

Matthew C. Winter1, Caroline Wilson1, Stuart P. Syddall1, Simon S. Cross2,

Alyson Evans1, Christine E. Ingram3, Ingrid J. Jolley3, Matthew Q. Hatton1,

Jennifer V. Freeman4, Stefano Mori1, Ingunn Holen1 and Robert E. Coleman1,5

1Academic Unit of Clinical Oncology, CR-UK/YCR Sheffield Cancer Research

Centre, Weston Park Hospital, Sheffield, UK 2Academic Unit of Pathology, Department of Neuroscience, Faculty of Medicine,

Dentistry and Health, University of Sheffield, Sheffield, UK 3Department of Radiology, Royal Hallamshire Hospital, Sheffield, UK 4School of Health and Related Research, University of Sheffield, Sheffield, UK 5Sheffield Experimental Cancer Medicine Centre, University of Sheffield, Sheffield,

UK

Running title: Neoadjuvant chemotherapy + zoledronic acid in breast cancer

Keywords: apoptosis, breast cancer, chemotherapy, neoadjuvant, zoledronic acid

Corresponding author:

Dr. Matthew C. Winter MB ChB MSc MD MRCP

Academic Unit of Clinical Oncology

CR-UK/YCR Sheffield Cancer Research Centre

Weston Park Hospital, Whitham Road, Sheffield, UK

email: [email protected]

Tel: +44 114 2265079 Fax: +44 114 2265678

Conflicts of Interests:

Dr M. C. Winter: Speaker fees from Novartis

Dr I. Holen: Research funding from Novartis

Professor R.E. Coleman: Expert testimony for Novartis, Speaker fees from Amgen

Word Count: Translational relevance n =150, Abstract = 238, Manuscript = 4,165

Total number of figures and tables: 6 (2 tables and 4 figures)

Research. on July 18, 2021. © 2013 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 20, 2013; DOI: 10.1158/1078-0432.CCR-12-3235

http://clincancerres.aacrjournals.org/


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Statement of translational relevance

Zoledronic acid (ZOL) is firmly established in the management of patients with

metastatic bone disease. Additionally, data from recent adjuvant breast cancer studies

suggest a disease-modifying role in post-menopausal (naturally or medically-induced)

patients. The potential for an anti-tumour effect of bisphosphonates in the early breast

cancer setting represents an exciting clinical strategy. Beneficial anti-tumour effects

may result from an indirect effect through the inhibition of osteolysis, or alternatively,

through direct anti-tumour effects. Pre-clinical in vivo studies have shown sequence-

dependent anti-tumour effects of the addition of ZOL to chemotherapy, including

increased tumour cell apoptosis, reduced tumour proliferation and inhibition of

tumour vascularisation, with maximum effects observed when ZOL is administered

24-hours after chemotherapy. This neoadjuvant study investigated the potential

clinical significance of these effects evaluating the biological effects of the addition of

ZOL to chemotherapy on the primary tumour, circulating surrogate markers of

angiogenesis and reproductive hormones within the TGFß family.





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ABSTRACT

Purpose: To investigate the short-term biological effects of neoadjuvant

chemotherapy +/- zoledronic acid (ZOL) in invasive breast cancer

Experimental design: Forty patients were randomised to receive a single 4mg

infusion of ZOL 24 hours after the first cycle of FE100C chemotherapy, or

chemotherapy alone. Randomisation was stratified for tumour stage, ER, HER2, and

menopausal status. All patients had repeat breast core-biopsy at Day 5 (D5) +/- Day

21 (D21). Effects on apoptotic index, proliferation (Ki67), growth index, surrogate

serum markers of angiogenesis (VEGF) and serum reproductive hormones within the

TGFβ family (activin-A, TGFβ1, inhibin-A and follistatin) were evaluated and

compared.

Results: Baseline clinico-pathological characteristics were well balanced. Cell

growth index (increased apoptosis and reduced proliferation) fell at D5 in both groups

but recovered more rapidly with chemotherapy+ZOL than chemotherapy alone by

D21 (p=0.006). At D5, a greater reduction in serum VEGF occurred with

chemotherapy+ZOL compared to chemotherapy: median percentage change -23.8%

(IQR -32.9, -15.8) vs. -8.4% (IQR -27.3, +8.9; p=0.02), but these effects were lost by

D21. Postmenopausal women demonstrated a decrease in follistatin levels from

baseline in the chemotherapy+ZOL group at D5 and D21, compared to chemotherapy

alone (interaction p=0.051).

Conclusions: In this pilot study, short-term changes in biomarkers suggest potentially

relevant interactions between tumour biology, chemotherapy, modification of the

bone microenvironment and the endocrine status of the host. Larger studies with more

frequent dosing of zoledronic acid are needed to assess these complex interactions

more thoroughly.





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Introduction

Zoledronic acid (ZOL) is firmly established in the management of patients with

metastatic bone disease [1] and may have a role in the adjuvant treatment of breast

cancer [2]. ZOL inhibits farnesyl diphosphate (FPP) synthase, a key enzyme in the

mevalonate pathway, ultimately leading to osteoclast apoptosis. There are also a

wealth of pre-clinical data reporting that ZOL-induced FPP synthase inhibition has

both direct and indirect anti-tumour effects in breast cancer [3]. Pre-clinical studies

have shown sequence-dependent synergy between chemotherapy agents and ZOL [4,

5] with maximum effects observed when ZOL is administered 24 hours after

chemotherapy. Six cycles of weekly treatment with clinically relevant doses of

doxorubicin followed 24 hours later by ZOL inhibited subcutaneous tumour growth in

an in vivo mouse model of breast cancer soft tissue disease in the absence of tumour-

associated bone disease [6]. The anti-tumour effects included increased tumour cell

apoptosis, reduced tumour proliferation and inhibition of tumour vascularisation [4].

The same treatment schedule has also been shown to prevent the development and

progression of spontaneous mammary tumours in the immunocompetent PyMT

mouse model, again in the absence of bone disease [7].

The potential for an anti-tumour effect of bisphosphonates in the early breast cancer

setting represents an exciting clinical strategy. Beneficial anti-tumour effects may

result from an ‘indirect’ effect through the inhibition of bone resorption and

consequent reduction in bone-derived growth factors, disrupting interactions between

cancer and bone cells and the creation of a less favourable bone microenvironment for

the survival of disseminated tumour cells. Alternatively, ‘direct’ anti-tumour effects,

such as induction of tumour cell apoptosis, inhibition of proliferation and anti-

angiogenesis may also be important, in addition to potential synergistic effects with

anti-cancer treatments [2-4].

The role of adjuvant bisphosphonates has been investigated in several large

randomised adjuvant phase III trials, with some highly promising results in certain

subsets of patients [8, 9], but no significant improvements in disease free (DFS) or

overall survival (OS) across unselected populations of early breast cancer patients

[10, 11]. However, in women who are postmenopausal, either naturally [8, 10, 11] or

induced by goserelin [9], a consistent improvement in both DFS and OS with





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administration of adjuvant bisphosphonates has emerged. The biological rationale for

these observations is an area of intense study [12, 13]. The interaction between

menopause and effects of bisphosphonates is suggested to be linked to the TGFβ

family of growth factors [13]. Activin and TGFβ may act as paracrine tumour

suppressors in early breast cancer, and the ovarian secreted hormone, inhibin, can

decrease their activity. Activin is further inhibited by the paracrine glycoprotein

follistatin. Inhibin declines with menopause, and is undetectable in postmenopausal

women, whereas follistatin is not affected by menopausal transition. Bone acts as a

reservoir for both activin and TGFß, and bisphosphonates, through inhibition of bone

resorption, can decrease their release from bone [14, 15]. The significance of this

local bone effect of bisphosphonates on serum levels of the TGFß family has not been

previously explored.

We have previously shown in an exploratory analysis that the addition of ZOL to

neoadjuvant chemotherapy may improve tumour response in the breast, with smaller

residual tumour burden in the resection specimen and an increased rate of

pathological complete response [16]. These results, coupled with the pre-clinical data

[4-6], suggest that low circulating concentrations of ZOL, when given in combination

with chemotherapy, may be sufficient to exert anti-tumour effects in peripheral tissue

in vivo, or, suppression of bone turnover may mediate effects on tumour growth

outside bone. Alternatively, ZOL may affect survival / trafficking of bone marrow

precursors to peripheral tumours [17].

The neoadjuvant treatment setting provides a unique opportunity to assess the in vivo

activity of novel therapies by investigating patterns of treatment-induced changes of

biological marker expression. Here we report the results of a randomised phase II

study designed to investigate short-term biological changes in apoptosis and

proliferation within tumour tissue, as well as changes in serum angiogenesis markers

and TGFβ family members following the addition of ZOL to neoadjuvant

chemotherapy in invasive breast cancer (ANZAC - EUDRACT number 2007-001526-

27, ClinicalTrials.gov Identifier NCT00525759).





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Materials and Methods Study Patients

To be eligible, patients had to be female with a histological diagnosis of invasive

breast cancer and scheduled to receive neoadjuvant anthracycline-based

chemotherapy, aged ≥ 18 years with a WHO performance status of 0-2, have a T2-T4

tumour with no evidence of metastatic disease, and be prepared to undergo additional

tumour biopsies for research. Exclusion criteria included concurrent treatment with

tamoxifen or an aromatase inhibitor, need for oral anticoagulants, exposure to

bisphosphonates within the last year, active dental problems including dental abscess

or osteonecrosis of the jaw and insufficient renal function (creatinine clearance

<40mls/min).

Randomisation and Treatment

After written informed consent, 40 patients were randomised in a 1:1 ratio to receive

neoadjuvant chemotherapy with 5-Fluorouracil 600mg/m2, epirubicin 100mg/m2,

cyclophosphamide 600mg/m2 (FEC) every 3 weeks, followed by 3 cycles of

docetaxel 100mg/m2 (D) every three weeks alone) (FEC-D chemotherapy alone n=20)

or the same FEC-D chemotherapy plus one infusion of ZOL (4mg intravenous

infusion over 15 minutes) administered on day 2, 24 hours after the first cycle of FEC

(chemotherapy+ZOL n=20) utilising a similar schedule of administration in patients

to that shown to be most effective in the preclinical studies [4]. Trastuzumab was

introduced alongside docetaxel in HER2 positive patients. The study consisted of an

18-week neoadjuvant chemotherapy phase, followed by surgery that marked the end

of the study. Following surgery, patients received further adjuvant treatment as

clinically indicated and underwent standard follow-up.

The study was approved by the South Yorkshire Local Research Ethics Committee,

and the trial performed in accordance with ICH GCP and the EU Directive.

Trial procedures and techniques

Tissue biomarkers

All patients consented to a repeat core biopsy on day 5 (D5, approximately 96 hours

after chemotherapy and 72 hours after zoledronic acid), plus the option of a further





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core biopsy on day 21 (D21), just before the second cycle of chemotherapy. Core

biopsies (2 x 14G) were performed under ultrasound guidance by radiologists. Biopsy

specimens were formalin-fixed and processed to paraffin.

The apoptotic index (AI), expressed as a percentage of apoptotic cells counted out of

the total number of tumour cells examined, was assessed on invasive tumour in the

initial diagnostic core biopsy, D5 and D21 core biopsies, and the operative specimen.

Apoptotic cells were evaluated using the TUNEL (terminal deoxyribonucleotidyl

tranferase-mediated dUTP nick end labelling) assay. Briefly, five micrometer sections

were dewaxed, endogenous peroxide activity blocked by 1% hydrogen peroxide and

the sections treated with 0.5% pepsin (pH=2, Sigma, P7012) for 30 minutes at 37ºC

followed by extensive washing in dH2O and then in Tris buffered saline (pH=7.6).

Sections were incubated in a reaction mixture containing 15 units TdT [Promega UK,

M1875], 0.5nmol biotin 16dUTP [Roche Diag. 1093070910], 5mM cobalt chloride

[Sigma, 232696]), washed in dH2O and x3 in PBS/1% tween and incubated with 100µl

HRP conjugated streptavidin (R+D, Y998) for 30 minutes at ambient temperature.

Colour was developed by incubating with diaminobenzidine (DAB) (ImmPACT kit,

Vector Labs) following manufacturer’s instructions. Paired biopsies (+/- day 21 +/-

surgical resection specimen) were stained on the same run, and positive and negative

controls were included with each batch of slides. Tumour proliferation was assessed

by immunohistochemical tumour cell expression of Ki67 antigen on the sections

described above, using MM1 monoclonal antibody (Cat no. VP-K542, Vector Labs)

and Elite Mouse kit (Vector Labs, PK6102) following the manufacturers instructions.

Stained slides were scanned using Aperio ScanScope GL System (scanning

magnification 20x, resolution 0.25µm / pixel). Areas of invasive carcinoma on the

digital image of the section were selected at random by a specialist histopathologist

(S.S.C) blinded to treatment allocation and timing of sample.

Apoptotic index was assessed by counting a total of 2,000 tumour cells at x20

magnification and is reported as the number of positive apoptotic cells as a

percentage. Areas of extensive necrosis were avoided. TUNEL stained apoptotic cells

were recorded as positive and any unstained cells that displayed classic apoptotic

morphology (condensed irregular nuclei or fragmented nuclei within cells that





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showed cytoplasmic withdrawal) were also included in the apoptotic index. Ki67

score was calculated as the number of positive stained cells and expressed as a

percentage of the total number of tumour cells counted. A total of 1000 tumour cells

were counted in each section at each time-point for each score at x20 magnification.

All sections were counted blinded to treatment allocation and time-point.

Blood samples

Blood samples were taken into a clot activator tube containing no additive at four

time-points: prior to first chemotherapy, D5, D21 prior to second cycle of

chemotherapy and on the day of surgery. Platelet level was also evaluated. Blood was

allowed to clot at ambient temperature for 30-60 minutes before centrifuging at 2000G

for 10 minutes at ambient temperature. Samples were stored at -80oC. Serum

VEGF165, activin-A, TGFβ1, follistatin and inhibin-A levels were assayed using a

quantitative sandwich ELISA processed according to manufacturers instructions

(DVE00, DAC00B, DFN00, DB100B R&D Systems; DSL-10-28100-1 Beckman

Coulter, respectively). Samples were analysed in duplicate, with a coefficient of

variation ≤10%. Minimum detection limits were <5pg/ml for inhibin A, 29pg/ml for

follistatin, 3.67 pg/ml for activin A, and 1.7 pg/ml for TGFβ1. For serum VEGF165, the

range of detection was 31.25 – 2000 pg/ml. Clinical menopausal status was confirmed

biochemically by follicle stimulating hormone measured on a COBAS E602

autoanalyser.

Statistical Methods

Patients were randomised using an electronic minimisation programme and stratified

for tumour stage, oestrogen receptor (ER) status, HER2 status and menopausal status.

The primary outcome measure was change in apoptotic index between diagnostic core

biopsy and D5 core biopsy. Changes from baseline to D21 and to surgery were also

investigated. Secondary endpoints included evaluation of changes in Ki67

proliferation from baseline to subsequent time-points, changes in growth index

reflecting a combined contribution of proliferation and apoptosis to changes in

growth, expressed as a ratio of Ki67/apoptosis. Changes in serum VEGF, activin-A,

TGFβ1, follistatin and inhibin-A, from baseline to subsequent time-points, were also

evaluated.





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Statistical analysis was carried out using SPSS (SPSS Inc, Chicago, USA). Apoptosis,

Ki67 and serum VEGF165 data were non-normally distributed and comparisons of

baseline and post-treatment samples (D5, D21 and surgery) for the patient group as a

whole were performed using Wilcoxon matched-pair signed-rank test. Differences in

change in AI, Ki67 and serum VEGF165 from baseline to subsequent time-points

between treatment groups were assessed using the Mann-Whitney test. To analyse a

treatment:menopause interaction for activin-A, TGFβ1, follistatin and inhibin-A,

differences between groups in median values, from baseline to subsequent time-

points, were assessed using a linear regression model.

The sample size was based on treatment-induced increase in apoptosis 24 hours after

chemotherapy reported by Archer et al. [18]. With a sample size of 20 patients in each

treatment arm, the study had 80% power to detect a 3.5 fold increase in apoptosis in

the chemotherapy-ZOL sequenced arm over and above the increase expected due to

chemotherapy alone. A 3.5 fold enhancement of apoptosis had been seen with the

chemotherapy-ZOL sequence in the preclinical animal model studies.4-6

Results Between July 2007 and July 2009, 40 patients were recruited (chemotherapy n=20,

chemotherapy+ZOL n=20). Baseline and post-treatment clinico-pathological

characteristics and treatment details are presented in Table 1. All patients underwent a

repeat core biopsy on D5. Figure 1 demonstrates patient flow through the study and

reports the number of evaluable and non-evaluable tumour biopsies for AI and Ki67

at each time-point for each treatment group.

The addition of ZOL to the first chemotherapy cycle was well tolerated with no

increase in serious adverse events reported. Four patients did not complete six cycles

of chemotherapy due to toxicity, including 3 patients proceeding to surgery after 4 or

5 cycles (Table 1). A further patient progressed following 3 chemotherapy cycles and

underwent surgery, and 2 patients did not have surgery (n=1 death due to pulmonary

embolus prior to surgery, n=1 received radiotherapy only).





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Apoptotic Index (AI)

Median baseline AI was 2.98% (inter-quartile range (IQR) 1.55, 4.45) in the

chemotherapy group and 2.35% (IQR 1.20, 3.48) in the chemotherapy+ZOL group.

For the group as a whole, there was a median absolute increase in AI at D5 of 0.7%,

(IQR +0.1, +3.6) p=0.003 (n=33). This effect was lost by D21, with a median absolute

decrease in AI of -0.15% (IQR -1.1, +0.3, p=0.21, n=20) between baseline and D21.

Between D5 and D21, there was a significant decrease in AI for the group as a whole

(median change -1.9% [IQR -4.8, -0.6], p=0.001, n=20). AI at surgery was

significantly lower than at baseline (median absolute change -0.5% [IQR -1.3, -0.2],

p<0.0001, n=20).

Between the treatment groups (Table 2), there was no significant difference in median

% change in AI from baseline to D5 (chemotherapy +81.4% [IQR -18.0, +102.2],

chemotherapy+ZOL +46.0% [IQR +7.8, +188.5], p=0.48), or from baseline to any

subsequent time-points.

Proliferation (Ki67)

Median baseline Ki67 was 25.7% (IQR 16.2, 50.7) in the chemotherapy group and

21.7% (IQR 13.2, 42.7) in the chemotherapy+ZOL group. For the whole group, there

was a non-significant reduction in Ki67 from baseline to D5 (absolute median

reduction in Ki67 of -7.1%, [IQR -15.9, +9.0, p=0.09], n=35), and a borderline

significant reduction from baseline to D21 (absolute median reduction in Ki67, -5.9%

[IQR -16.8, 1.7], p=0.045, n=23). There was a significant reduction in Ki67 between

baseline and surgery (median absolute decrease in Ki67: -13.0% [IQR -19.4, -7.7],

p=0.005).

Between the treatment groups, there was no difference in Ki67 decrease between

baseline and D5 (median % change in Ki67: -18.4% [IQR -58.9, +20.6] in the CT

group and -24.2% [IQR -91.0, +17.5] in the chemotherapy+ZOL group, p=0.44).

However, by D21 there was significant recovery of Ki67 in the chemotherapy+ZOL

group to levels above baseline (median Ki67 change: +184.8% [IQR 3.9, 331.67]), an

effect not seen in the chemotherapy group (median change Ki67: -26.9% [IQR -45.7,

-1.7], p=0.009). This dominant effect is also seen in the evaluation of change in

growth index (Table 2 and Figures 2a-c).





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Vascular Endothelial Growth Factor (VEGF)

Baseline serum VEGF level was evaluable in 39 patients (chemotherapy n=20,

chemotherapy+ZOL n=19). One patient was excluded from analysis as serum VEGF

level was below the lower limit of detection at all time-points. In another patient

receiving chemotherapy+ZOL, the serum VEGF level was below the lower limit of

detection at D5 and was allocated a value of 31.25pg/ml. In one patient receiving

chemotherapy alone, serum VEGF level was above the upper limit at D21 and was

therefore allocated an upper limit of detection value of 2000pg/ml. Serum VEGF at

surgery was evaluable in 26 patients (n=13 in each group).

Median baseline serum VEGF in patients receiving chemotherapy alone was

354.1pg/ml (IQR 170.7, 511.2), and 352.0 pg/ml (240.3, 627.6) in patients receiving

chemotherapy+ZOL. All patients receiving chemotherapy+ZOL demonstrated a

decrease in serum VEGF at D5. Eight (40%) patients in the CT alone group

demonstrated a rise in serum VEGF at D5 (Figure 3a). Median percentage reduction

in serum VEGF from baseline to D5 in the chemotherapy alone group was -8.4%

(IQR: -27.3, +8.9) compared to -23.8% (IQR: -32.9, -15.8) in the chemotherapy+ZOL

group (p=0.024) (Table 2). Following the decrease in serum VEGF at D5, there was a

rebound increase in serum VEGF in both treatment groups to levels above baseline

(Figure 3b), likely to be associated with platelet recovery following chemotherapy.

However there was no difference between the treatment groups in change in serum

VEGF from baseline to D21 or to surgery. Importantly, there was no significant

difference between the two groups in change in quantitative platelet value between

the two groups from baseline to D5 or to any other time-point, although there was a

clear increase in platelet count from D5 to D21 in both groups (data not shown).

Reproductive hormones and TGFβ family

Figures 4a-b shows the median and IQR values for follistatin at baseline, D5 and D21

by treatment group and menopausal status. Baseline values were similar in all four

groups. The changes with time with or without ZOL appear to be influenced by

menopausal status. In the post-menopausal group, median follistatin levels fell from a

baseline median of 1596pg/ml in the chemotherapy+ZOL group at both day 5





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(1102pg/ml) and day 21 (1085pg/ml), compared to an increase from 1454pg/ml in the

chemotherapy group at both day 5 (1544pg/ml) and day 21 (1689pg/ml). Changes in

premenopausal patients were similar in both treatment groups. Using a linear

regression model, an interaction between menopausal status and treatment group of

borderline significance was demonstrated (p=0.051), suggesting that the addition of

ZOL to chemotherapy differentially affects serum follistatin dependent on the

menopausal status of the patient.

No significant interaction between menopausal status and treatment group was

demonstrated for activin A or TGFβ1. As expected, menopausal status significantly

influenced inhibin-A levels, with much lower levels in post-menopausal women

(mean difference in AUC (pre-post): 16.9 pg/ml, 95% CI: 9.1, 24.6, P<0.001), but

these were not affected by treatment received (Figure 4c-d).

Discussion

In this pilot study, we investigated the short-term biological effects of the addition of

ZOL to the first cycle of chemotherapy, using the same schedule that demonstrated

synergy in preclinical experiments [4-6], albeit only for a single cycle as compared to

weekly treatments for 6 cycles in the pre-clinical setting. In this study, no definite

evidence of a direct anti-tumour effect of the addition of ZOL to CT can be

concluded, but potentially relevant biological effects were seen, and a possible novel

interaction between ZOL and menopausal status demonstrated within the endogenous

inhibitors of the TGFβ family of growth factors.

Chemotherapy induced a statistically significant increase in apoptotic index from

baseline to D5. However, there were no differences in apoptotic index between

treatment groups at any time-point, suggesting that ZOL had no additional effect on

the induction of apoptosis by chemotherapy at the primary site. The optimal timing of

subsequent biopsy to capture treatment-induced increases in apoptosis is unknown

and this does represent a limitation of any breast cancer biomarker study assessing

apoptosis as an end-point.

There are only a few studies that have evaluated apoptosis changes during

neoadjuvant chemotherapy for breast cancer and the majority of these assessed





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apoptosis after 24-72 hours [18-22]. Arpino et al. measured apoptosis (using TUNEL)

and Ki67 at baseline, at 24-48 hours and on day 7 (D7) after single agent

anthracycline chemotherapy [19]. From baseline to day 1, absolute median apoptosis

increased by 0.6% (n=13 paired samples only). In studies by Ellis [22] and Archer

[18] respectively, absolute median apoptotic index increased by 0.55% and 0.7%

respectively at 24 hours following anthracycline-based chemotherapy. Arpino et al.

[19] also evaluated apoptosis at D7 showing a median increase of 0.1% from baseline,

suggesting that chemotherapy-induced effect on apoptosis has waned by day 7 and is

too late for optimal assessment.

Our study demonstrates a significant chemotherapy-induced increase in apoptotic

index that is sustained to at least D5, with an absolute median increase in apoptotic

index of 0.7% for the whole group. It is possible that day 5 is too late and any early

anti-tumour effects of ZOL may have been missed and, in addition, the time of peak

increase in apoptosis may be variable between patients. In the pre-clinical studies

reporting increased levels of tumour cell apoptosis following the addition of ZOL to

chemotherapy, tumours were always collected 24 hours after the last of 6 weekly

injections of ZOL [4, 6, 7]. Following a 4mg intravenous dose of ZOL administered

over 15 minutes, there is a sharp increase in concentration with a peak plasma

concentration after the end of infusion of approximately 1μM [23] followed by a

rapid decline in plasma concentration with prolonged (terminal elimination half-life

approximately 7 days) but very low drug plasma concentrations thereafter.

A decrease in tumour cell proliferation has been shown to occur as early as 24-48

hours after chemotherapy [18-20, 22]. In our study, for the group as a whole, there

was an absolute median reduction of -7.1% (IQR -15.9, 9.0) in the number of Ki67

positive cells between baseline and D5 and similar in magnitude to other reports.

However, our study did not show any beneficial anti-proliferative effect of the

addition of ZOL to chemotherapy at this early time point.

Interestingly, significant differences in Ki67 between groups were seen at day 21.

From D5 to D21 there was recovery of proliferation to levels above baseline in

patients in the chemotherapy+ZOL group but not in the chemotherapy alone group.

These results are likely to be driven by a small number of patients in the





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chemotherapy+ZOL group that demonstrated large rebound increases in Ki67 to D21.

However, further evaluation of this phenomenon and of the biological and clinical

consequences of a rebound in proliferation prior to subsequent chemotherapy merit

investigation.

A wide variation between individual patients in AI and Ki67 was seen with no

consistent pattern across all patients with respect to change from baseline to

subsequent time-points. This implies there is more variation between individual

patients than between treatment groups and, although our study stratified patients in

an attempt to account for biological factors such as oestrogen receptor status that

influence chemo-sensitivity, a larger study is needed to reliably evaluate treatment

interactions across biological subtypes.

Despite ZOL localizing quickly to bone, pre-clinical evidence suggests that increased

penetration of ZOL into extra-skeletal tumour tissue occurs following exposure to

doxorubicin. Using in vivo subcutaneous MDA-MB-436 tumours, Ottewell et al.

reported that in tumours treated sequentially with doxorubicin followed by ZOL

weekly for 6 weeks, unprenylated Rap1a could be detected by Western blot analysis,

and not detected in tumours following the reverse sequence or either drug alone [6].

We found that the addition of ZOL to chemotherapy causes a short-term reduction in

VEGF compared to chemotherapy alone. This appears to be independent of platelet-

VEGF contribution, as we found no significant difference in quantitative platelet

count between the treatment groups from baseline to D5. This possible effect of ZOL

is of potential clinical significance, as studies have correlated elevated circulating

levels of VEGF with disease progression or poor survival [24]. However, any

potential beneficial anti-angiogenic effect was lost by 21 days, and a single infusion

of ZOL is unable to suppress the rebound increase in serum VEGF observed in both

groups at D21, likely explained by platelet recovery following chemotherapy [25]. A

repeated low-dose schedule of ZOL has been shown to induce a longer lasting

decrease in circulating VEGF levels [26], suggesting that a more frequent dosing

schedule is required to exploit any potential anti-angiogenic effects of ZOL.





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The mechanism of a short-term beneficial effect is unclear. Circulating levels of

VEGF have been correlated with levels of circulating endothelial cells in cancer

patients [27], but not in other studies [28]. Hasmim et al. demonstrated ZOL-induced

inhibition of human umbilical vein endothelial cell adhesion, survival and migration

by interfering with protein prenylation in several signalling pathways [29]. Giraudo et

al. demonstrated in a mouse cervical cancer model that ZOL suppressed expression of

matrix metalloproteinase-9 and reduced the association of VEGF with its receptor on

angiogenic endothelial cells [30].

Our data are the first to report that addition of ZOL to chemotherapy in

postmenopausal women decreases serum levels of the activin inhibitor, follistatin,

compared to an increase with chemotherapy alone. This occurs in a group of patients

with concurrently low levels of the alternative activin inhibitor, inhibin A. In

addition, quantitative serum levels of activin and TGFß were not significantly altered

by ZOL, suggesting that that the microenvironment of potential extra-osseous sites of

micro-metastatic tumour growth may not be subject to lower levels of tumour

suppressor growth factors.

A decrease in follistatin with ZOL may act to increase the biological availability of

activin in the local tumour environment, thus promoting the tumour suppressor action

of this TGFb family member, with the resultant effect that a postmenopausal female

receiving ZOL may have a higher biological availability of activin compared to a

premenopausal woman. Changes in serum levels of follistatin may not entirely reflect

changes in the intratumoural levels of these paracrine factors, however modification

of pre-metastatic niches may be a proposed mechanism by which serum levels can

alter survival of disseminated tumour cells at distant sites. This may potentially

contribute to the beneficial effects on extra-osseous recurrence rates seen in the

clinical trials in women with a natural or induced menopause [8-11]. These complex

interactions require further research, both in vitro and in vivo, to identify the link

between menopause and the response to ZOL.

In summary, no definite evidence of a direct anti-tumour effect of the addition of ZOL

to chemotherapy was observed although ZOL may have transient anti-angiogenic

potential outside of bone. The single dosing of ZOL in our study is a limitation, and it





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is likely in the clinical setting that a more frequent dosing with chemotherapy is

required to demonstrate any clinically relevant direct anti-tumour effect. The

biological significance of recovery of tumour cell proliferation by D21 in patients

treated with chemotherapy+ZOL and the differential effects by menopause on

circulating follistatin levels warrant further study in view of the emerging clinical trial

results [2].

Acknowledgements:

We would like to acknowledge Janet Horsman, Cancer Clinical Trials Centre,

University of Sheffield; Yvonne Stephenson and team, Medical School, University of

Sheffield; Simone Detre, Institute of Cancer Research, Royal Marsden, London;

Rosie Taylor, School of Health and Related Research, University of Sheffield and

Weston Park Hospital Cancer Charity for part funding of this research.





17

References 1. Coleman RE, McCloskey EV. Bisphosphonates in oncology. Bone. 2011;

49(1): 71-6. 2. Coleman RE, Gnant M, Morgan G, Clezardin P. Effects of bone-targeted

agents on cancer progression and mortality. J Natl Cancer Inst. 2012; 104 (14): 1059-67.

3. Winter MC, Holen I, Coleman RE. Exploring the anti-tumour activity of bisphosphonates in early breast cancer. Cancer Treat Rev. 2008; 34(5): 453-75.

4. Ottewell PD, Monkkonen H, Jones M, Lefley DV, Coleman RE, Holen I. Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer. J Natl Cancer Inst. 2008; 100(16): 1167-78.

5. Ottewell PD, Woodward JK, Lefley DV, Evans CA, Coleman RE, Holen I. Anticancer mechanisms of doxorubicin and zoledronic acid in breast cancer tumor growth in bone. Mol Cancer Ther. 2009; 8(10): 2821-32.

6. Ottewell PD, Lefley DV, Cross SS, Evans CA, Coleman RE, Holen I. Sustained inhibition of tumor growth and prolonged survival following sequential administration of doxorubicin and zoledronic acid in a breast cancer model. Int J Cancer. 2010; 126 (2): 522-532.

7. Ottewell PD, Brown HK, Jones M, Rogers TL, Cross SS, Brown NJ, et al. Combination therapy inhibits development and progression of mammary tumours in immunocompetent mice. Breast Cancer Res Treat. 2011; 133(2): 523-36.

8. de Boer R, Bundred N, Eidtmann H, Neven P, von Minckwitz G, Martin N, et al. Long-term survival outcome among post-menopausal women with hormone receptor-positive early bresat cancer receiving adjuvant letrozole and zoledronic acid; 5-year follow-up of ZO-FAST. Cancer Research. 2011; 71 (24 Suppl): Abstract S1-3.

9. Gnant M, Mlineritsch B, Schippinger W, Luschin-Ebengreuth G, Postlberger S, Menzel C, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N Engl J Med. 2009; 360(7): 679-91.

10. Coleman RE, Marshall H, Cameron D, Dodwell D, Burkinshaw R, Keane M, et al. Breast-cancer adjuvant therapy with zoledronic acid. N Engl J Med. 2011; 365(15): 1396-405.

11. Paterson AH, Anderson SJ, Lembersky BC, Fehrenbacher L, Falkson CI, King KM, et al. Oral clodronate for adjuvant treatment of operable breast cancer (National Surgical Adjuvant Breast and Bowel Project protocol B-34): a multicentre, placebo-controlled, randomised trial. Lancet Oncol. 2012; 13(7): 734-42.

12. Hadji P, Coleman R, Gnant M, Green J. The impact of menopause on bone, zoledronic acid, and implications for breast cancer growth and metastasis. Ann Oncol. 2012; 23 (11): 2782-2790.

13. Wilson C, Holen I, Coleman RE. Seed, soil and secreted hormones: Potential interactions of breast cancer cells with their endocrine/paracrine microenvironment and implications for treatment with bisphosphonates. Cancer Treat Rev. 2012; 38(7): 877-89.

14. Korpal M, Yan J, Lu X, Xu S, Lerit DA, Kang Y. Imaging transforming growth factor-beta signaling dynamics and therapeutic response in breast cancer bone metastasis. Nat Med. 2009; 15(8): 960-6.





18

15. Sakai R, Eto Y, Hirafuji M, Shinoda H. Activin release from bone coupled to bone resorption in organ culture of neonatal mouse calvaria. Bone. 2000; 26(3): 235-40.

16. Coleman RE, Winter MC, Cameron D, Bell R, Dodwell D, Keane MM, et al. The effects of adding zoledronic acid to neoadjuvant chemotherapy on tumour response: exploratory evidence for direct anti-tumour activity in breast cancer. Br J Cancer. 2010; 102(7): 1099-105.

17. Gallo M, De Luca A, Lamura L, Normanno N. Zoledronic acid blocks the interaction between mesenchymal stem cells and breast cancer cells: implications for adjuvant therapy of breast cancer. Ann Oncol. 2011; 23(3): 597-604.

18. Archer CD, Parton M, Smith IE, Ellis PA, Salter J, Ashley S, et al. Early changes in apoptosis and proliferation following primary chemotherapy for breast cancer. Br J Cancer. 2003; 89: 1035-1041.

19. Arpino G, Ciocca DR, Weiss H, Allred DC, Daguerre P, Vargas-Roig L, et al. Predictive value of apoptosis, proliferation, HER-2, and topoisomerase II alpha for anthracycline chemotherapy in locally advanced breast cancer. Breast Cancer Res Treat. 2005; 92(1): 69-75.

20. Chang J, Ormerod M, Powles TJ, Allred DC, Ashley SE, Dowsett M. Apoptosis and Proliferation as Predictors of Chemotherapy Response in Patients with Breast Carcinoma. Cancer. 2000; 89: 2145-52.

21. Davis DW, Buchholz TA, Hess KR, Sahin AA, Valero V, McConkey DJ. Automated Quantification of apoptosis after neoadjuvant chemotherapy for breast cancer: early assessment predicts clinical response. Clinical Cancer Research. 2003; 9: 955-960.

22. Ellis PA, Smith IE, McCarthy K, Detre S, Salter J, Dowsett M. Preoperative chemotherapy induces apoptosis in early breast cancer. Lancet. 1997; 349(9055): 849.

23. Weiss HM, Pfaar U, Schweitzer A, Wiegand H, Skerjanec A, Schran H. Biodistribution and plasma protein binding of zoledronic acid. Drug Metab Dispos. 2008; 36(10): 2043-9.

24. Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol. 2001; 19(4): 1207-25.

25. Verheul HM, Hoekman K, Luykx-de Bakker S, Eekman CA, Folman CC, Broxterman HJ, et al. Platelet: transporter of vascular endothelial growth factor. Clin Cancer Res. 1997; 3(12 Pt 1): 2187-90.

26. Santini D, Vincenzi B, Galluzzo S, Battistoni F, Rocci L, Venditti O, et al. Repeated intermittent low-dose therapy with zoledronic acid induces an early, sustained, and long-lasting decrease of peripheral vascular endothelial growth factor levels in cancer patients. Clin Cancer Res. 2007; 13(15 Pt 1): 4482-6.

27. Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini F. Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood. 2001; 97(11): 3658-61.

28. Furstenberger G, von Moos R, Lucas R, Thurlimann B, Senn HJ, Hamacher J, et al. Circulating endothelial cells and angiogenic serum factors during neoadjuvant chemotherapy of primary breast cancer. Br J Cancer. 2006; 94(4): 524-31.

29. Hasmim M, Bieler G, Ruegg C. Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways. J Thromb Haemost. 2007; 5(1): 166-73.





19

30. Giraudo E, Inoue M, Hanahan D. An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest. 2004; 114(5): 623-33.





20

Legends to Figures Figure 1: CONSORT diagram to show patient flow through study

Figure 2a: Waterfall plot of percentage change in Growth Index from baseline to day

5 per patient per treatment group

Figure 2b: Waterfall plot of percentage change in Growth Index from baseline to day

21 per patient per treatment group

Figure 2c: Figure 3c: Growth Index (Ki67/AI) measured at baseline, day 5, day 21

and surgery shown per patient and by treatment group

Figure 3a: Waterfall plot of percentage change in serum VEGF from baseline to day 5

per patient per treatment group

Figure 3b: Serum VEGF measured at baseline, day 5, day 21 and surgery shown per

patient and by treatment group

Figure 4: Levels of serum follistatin (a and b) and inhibin (c and d) over time

according to menopausal status and treatment received




Table 1: Baseline and post-treatment clinico-pathological characteristics and treatment for the ANZAC study CT alone

n=20 (%)

CT + ZOL

n=20 (%) Median age, years (IQR) 49 (46 – 57)

51 (46 – 56)

Tumour stage T2-T3 T4 T4d

17 (85.0) 1 (5.0) 2 (10.0)

16 (80.0)

1 (5.0) 3 (15.0)

Menopausal status Pre Post

11 (55.0) 9 (45.0)

11 (55.0) 9 (45.0)

ER status* Positive Negative

14 (70.0) 6 (30.0)

16 (80.0) 4 (20.0)

HER2 status** Positive Negative

8 (60.0) 12 (40.0)

9 (45.0) 11 (55.0)

Chemotherapy treatment Completed 6 cycles Completed 5 cycles only Completed 4 cycles only Completed 3 cycles only Zoledronic acid with 1st cycle of FE100C chemotherapy

17 (85.0) 1 (5.0) 1 (5.0) 1 (5.0)

-

19 (95.0)

1 (5.0) - -

20 (100.0)

No. of patients undergoing Day 5 (D5) biopsy Day 21 (D21) biopsy

20 (100.0) 14 (70.0)

20 (100.0) 14 (70.)

Post-treatment pathological response at surgery Residual invasive disease in breast pCR (breast only) pCR (breast and axilla) No surgery Died prior to surgery

13 (65) 2 (10) 3 (15) 1 (5) 1(5)

13 (65) 3 (15) 4 (20) 0 (0) 0 (0)

*ER status: positive defined as McCarty’s H score ≥ 50 **HER2 status: positive defined by IHC = 3 or FISH amplified




Table 2: Change from baseline pre-treatment levels to subsequent time-points in AI, Ki67, Growth Index and serum VEGF165

Factor Time-point B: baseline D5: Day 5 D21: Day 21 Surgery

Median Percentage Change (%) Median Absolute change

(IQR)

P value

CT [n] CT+ZOL [n] Apoptotic Index (AI)

B – D5 B – D21 D5 – D21 B – Surgery

+81.4 (-18.0, 102.2) [17]

+0.9 (-0.8, 4.8) - 12.9 (-27.9, 29.5) [11]

-0.2 (-0.8, 0.5) - 56.9 (-61.0, -26.2) [11]

-1.8 (-4.8, -0.5) - 40.8 (-55.6, -35.3) [10]

-1.1 (-1.4, -0.6)

+46.0 (7.8, 188.5) [16]

+0.7 (0.2, 1.5) - 14.3 (-65.2, -2.2) [9]

-0.2 (-1.2, -0.1) - 61.1 (-70.7, -47.1) [9]

-2.0 (-3.0, -0.7) - 18.2 (-42.9, -13.6) [10]

-0.4 (-0.5, -0.2)

0.48 0.85 0.41 0.55 0.30 0.82 0.08 0.06

Ki67


- 18.4 (-58.9, 20.6) [19]

-7.4 (-16.6, 10.1) - 44.9 (-63.6, -17.1) [13]

-15.3 (-25.6, -3.7) - 26.9 (-45.7, -1.7) [13]

-7.8, (-14.5, -0.3) - 36.5 (-89.1, -24.0) [11]

-9.1 (-17.2, -7.1)

- 24.2 (-91.0, 17.5) [16]

-5.5 (-13.4, 6.9) +12.7 (-18.9, 47.7) [10]

+1.7 (-6.6, 7.4) +184.8 (3.9, 331.6) [9]

+6.3 (1.7, 19.0) - 55.8 (-74.5, -27.2) [10]

-14.0 (-23.2, -9.4)

0.44 0.96 *0.003 *0.01 *0.009 *0.001 0.86 0.39

Growth Index (KI67/AI)


- 35.6 (-56.0, 2.2) [17]

-3.1 (-10.9, 0.1) - 36.7 (-69.1, -0.1) [11]

-5.0 (-10.6, 0.0) + 24.2 (10.1, 124.1) [11]

+1.9 (0.8, 2.7) - 19.5 (-88.5, 22.6) [10]

-2.9 (-11.3, 2.0)

- 68.4 (-94.4, -15.8) [16]

-5.8 (-12.3, -3.4) + 72.4 (35.9, 152.9) [9]

+10.7 (5.5, 28.6) + 405.4 (214.0, 1234.0) [9]

+23.0 (9.9, 54.6) - 40.5 (-69.5, -14.3) [10]

-6.9 (-17.5, -1.2)

0.11 0.96 *0.002 *0.002 *0.006 <0.0010.97 0.28

Serum VEGF165


- 8.4 (-27.3, 8.9) [20]

-16.8 (-98.6, 20.1) + 59.3 (19.1, 97.8) [20]

+189.3 (74.7, 314.7) + 81.3 (16.6, 162.4) [20]

+202.7 (54.4, 397.3) + 19.6 (-13.4, 32.5) [13]

+72.5 (-53.1, 98.9)

- 23.8 (-32.9, -15.8) [19]

-73.6 (-103.9, -48.4) + 39.1 (28.7, 102.7) [19]

+183.3 (97.3, 296.2) + 100.5 (70.3, 178.5) [19]

+272.3 (201.2, 416.9) + 4.4 (-15.1, 24.9) [13]

+14.7 (-70.7, 91.6)

*0.02 *0.035 0.90 0.90 0.24 0.23 0.61 0.45

Figure in [brackets] denotes number of paired samples available for analysis *statistically significant at p<0.05




Figure 1: CONSORT diagram to show patient flow through study

Key: CT = chemotherapy, ZOL = zoledronic acid, AI = Apoptotic Index, DCIS =

ductal carcinoma in situ, pCR = pathological complete response

ASSESSED FOR ELIGIBILITY n=83

R A N D O M I S E D n = 40

CT ALONE n=20 CT + ZOL n=20

BASELINE AI assessable in 18 pts

(exhaustion of tumour block n=1,

insufficient invasive tumour n=1)

Ki67 assessable in 19 pts

(insufficient invasive tumour n=1)

Not randomised n=43

9 pts opted for 1o surgery

14 pts other reasons (social /

transport problems)

4 pts not approached

6 pts screening failures

8 pts refused due to need for

extra biopsy

2 pts refused any treatment

DAY 5 AI assessable in 19 pts

(insufficient invasive tumour n=1)


BASELINE AI assessable in 19 pts

(exhaustion of tumour block n=1)


(exhaustion of tumour block n=1)


(declined biopsy n=6, DCIS only

n=1, insufficient invasive tumour

n=1)


(declined 2nd biopsy n=6, DCIS only

n=1)


(failed biopsy n=2, insufficient invasive

tumour n=2)

Ki67 in 16 pts (reasons as above)


(declined biopsy n=6, failed biopsy

n=2, insufficient invasive tumour n=2)


(declined biopsy n=6, failed biopsy

n=2, insufficient invasive tumour n=1)

SURGERY AI assessable in 12 pts

(pCR n=5, early surgery n=1, died

prior to surgery n=1, no surgery n=1)


(reasons as above)

SURGERY AI assessable in 10 pts

(pCR n=7, tumour blocks not available

n=2, no tumour in selected block n=1) Ki67 assessable in 10 pts

(reasons as above)




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Published OnlineFirst March 20, 2013.Clin Cancer Res Matthew C Winter, Caroline Wilson, Stuart P Syddall, et al. early breast cancer - a randomised biomarker pilot studyNeoadjuvant chemotherapy with or without zoledronic acid in

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