Antitumoral Activiti of HER2

7/22/2019 Antitumoral Activiti of HER2

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J Cancer Res Clin Oncol (2009) 135:13771386

DOI 10.1007/s00432-009-0579-3

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ORIGINAL PAPER

Antitumor activity of the HER2 dimerization inhibitor

pertuzumab on human colon cancer cells in vitro and in vivo

Michael Pohl I. Stricker A. Schoeneck K. Schulmann S. Klein-Scory

I. Schwarte-WaldhoV M. Hasmann A. Tannapfel W. Schmiegel

A. Reinacher-Schick

Received: 13 July 2008 / Accepted: 16 March 2009 / Published online: 2 April 2009

Springer-Verlag 2009

Abstract

Purpose The monoclonal antibody pertuzumab represents

the Wrst HER2 dimerization inhibitor with unknown activ-

ity in colon cancer treatment. We examined the antitumor

activity of pertuzumab as a single agent or in combination

with erlotinib or irinotecan in human colon cancer cells in

vitro and in vivo.

Methods Colon cancer cell lines were tested for HER1/

HER2 expression by western blot analysis. The eVect of

pertuzumab on cell cycle distribution was analyzed by

FACS. Nude mice bearing xenograft tumors were treated

with pertuzumab alone, or in combination either with irino-

tecan or with erlotinib. Tumor volume was measured

repeatedly. Tumor histology was analyzed for necrosis.

Results Six of nine cell lines showed high expression of

HER1/HER2. Pertuzumab inhibited cell cycle progression

in various cell lines. Pertuzumab showed minor antitumor

activity in xenograft tumors, but signiWcantly inhibited

tumor growth when combined with erlotinib (P < 0.001).

Combination of pertuzumab with irinotecan had no addi-

tional eVect on growth of additional tumors. Pertuzumab

treated DLD-1 xenograft tumors did not show enhanced

necrosis, which, however, was found in HCT116 derived

xenografts.

Conclusions Pertuzumab has some antitumor activity on

human colon cancer cells in vitro and in vivo, in particular

when combined with erlotinib. In vivo, pertuzumab combi-

nation treatment was not superior to irinotecan mono-

therapy. These data warrant further investigation of

simultaneous HER1/EGFR TKI inhibition and HER1/

HER2 dimerization inhibition for colorectal cancer therapy.

Keywords Colorectal cancer Pertuzumab HER

dimerization inhibitor Dual ErbB inhibition Irinotecan

Abbreviations

HER Human epidermal growth factor receptor

EGFR Epidermal growth factor receptor

MAPK Mitogen-activated protein kinase

TKI Tyrosine kinase inhibitor

Introduction

Colorectal cancer constitutes the second leading cause of

death from cancer in Europe and North America and is

responsible for approximately one million new cases and

half a million deaths per year worldwide (Ferlay et al.

2001). The human epidermal growth factor receptor

(EGFR, HER1, ErbB-1) belongs to the ErbB or HER

family of receptor tyrosine kinases and is overexpressed or

dysregulated in many gastrointestinal cancer types, includ-

ing 6080% of colon cancers, which in turn correlates with

M. Pohl (&) A. Schoeneck K. Schulmann S. Klein-Scory

I. Schwarte-WaldhoV W. Schmiegel A. Reinacher-Schick

Department of Medicine, Knappschaftskrankenhaus,

Ruhr University, In der Schornau 23-25, 44892 Bochum, Germany

e-mail: [email protected]

I. Stricker A. TannapfelInstitute of Pathology, BG University Clinics Bergmannsheil,

Ruhr University, Brkle-de-la-Camp-Platz 1, 44789 Bochum,

Germany

M. Hasmann

Roche Diagnostics GmbH, Nonnenwald 2,

82372 Penzberg, Germany

W. Schmiegel

Department of Gastroenterology and Hepatology,

BG University Clinics Bergmannsheil, Ruhr University,

Brkle-de-la-Camp-Platz 1, 44789 Bochum, Germany


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1378 J Cancer Res Clin Oncol (2009) 135:13771386

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poor prognosis and early disease progression (Hemming

et al. 1992; Mayer et al. 1993; Porebska et al. 2000;

Salomon et al. 1995). HER2 (ErbB-2) overexpression has

been previously reported in up to 85% of colon cancers;

however, estimates vary depending on the methods used

(Dursun et al. 2001; Ross and McKenna 2001). HER2

overexpression is associated with poor prognosis in some

studies (Kapitanovic et al. 1994, 1997), but few studieshave examined the therapeutic potential of HER2 inhibition

in colon cancers (Mann et al. 2001). In contrast, the HER1/

EGFR has been established to be an important therapeutic

target in colon and other human cancers.

In addition to HER1/EGFR and HER2, the HER family

of receptor tyrosine kinases (TK) includes HER3 (ErbB-3)

and HER4 (ErbB-4) (Carpenter and Cohen 1990; Real et al.

1986). They are usually activated by ligand binding to the

receptors and subsequent dimerization. However, there are

two exceptions: there is no ligand for HER2 and the TK

domain of HER3 is inactive. When HER3 is stimulated by

its ligands heregulin-1 or -2, it preferentially forms hetero-

dimers with HER2. The HER2/HER3 heterodimer is

known to provide a potent oncogenic stimulus by activating

both, proliferation and survival signaling cascades

(Mendelsohn 2002), such as the Ras/Raf/MEK (mitogen-

activated protein kinase or MAPK) and PI3 kinase/Akt/

mTOR signaling pathways (Baselga and Arteaga 2005).

Both HER1/EGFR and HER2 are co-expressed in colorectal

cancer cells and simultaneous targeting of these receptors

enhanced apoptosis in preclinical studies (Fields et al.

2005; Zhou et al. 2006).

Pertuzumab (rhu MAb 2C4; Genentech Inc., San Francisco,

CA) is the Wrst in the new class of targeted therapeutic agents

known as HER2 dimerization inhibitors (Adams et al. 2006).

Pertuzumab is a recombinant, humanized monoclonal anti-

body that speciWcally binds to an epitope on the dimerization

domain of HER2 (Franklin et al. 2004). Binding of pert-

uzumab prevents HER2 homo- and heterodimerization

(Agus et al. 2002). Because of this mechanism of action,

pertuzumab antitumor activity is not restricted to tumors with

HER2 overexpression and therefore diVers from the thera-

peutic monoclonal antibody trastuzumab (Herceptin,

Genentech Inc., San Francisco, CA), which binds to a non-

overlapping juxtamembrane region of HER2s extracellular

domain, cannot inhibit HER2 heterodimerization and requires

HER2 overexpression for antitumor activity (Friess et al.

2002; Cobleigh et al. 1999). Pertuzumab is also active in

low-to-moderate HER2 expressing ovarian and breast cancer

cell lines. Preclinical and early phase I/II activities have been

identiWed in several cancer types, including breast and ovar-

ian cancer (Agus et al. 2002, 2005; Jackson et al. 2004).

The topoisomerase I inhibitor irinotecan (CPT-11) is a

water soluble, semisynthetic derivative of camptothecin that

has shown activity against a number of diVerent tumor types

in preclinical models and in clinical trials of patients with vari-

ous human cancers (Saijo 2000). Despite clinical improve-

ments attributed to the addition of CPT-11 therapy for

metastatic colorectal cancer, nearly all patients will become

refractory to CPT-11. Thus, new treatment options are needed

to improve survival in patients with CPT-11 refractory colo-

rectal cancer. Cetuximab (Erbitux, Imclone, New York,

NY), a chimeric monoclonal antibody that blocks ligand-dependent EGFR receptor activation, is highly synergistic

with irinotecan in refractory disease of colorectal cancer

patients (Saltz et al. 2001, 2004). Cetuximab is FDA approved

for second line therapy in combination therapy and will soon

aquire Wrst line approval in combination with chemotherapy.

Lastly, erlotinib (OSI-774, Tarceva, Genentech Inc., San

Francisco, CA) is a selective, orally available low molecular

weight inhibitor that binds competitively to the ATP-binding

site at the kinase domain of EGFR. Preclinical studies with

erlotinib have shown potent antitumor activity in a variety of

cultured tumor cell lines as well as human colon cancer xeno-

graft tumors (Akita and Sliwkowski 2003; Grunwald and

Hidalgo 2003). Favorable clinical studies have led to the

approval of erlotinib for use in the treatment of advanced

non-small cell lung cancer (NSCLC) and advanced pancreatic

cancer (Johnson et al. 2005; Moore et al. 2007). Erlotinib in

colorectal cancer remains investigational. A phase II study

presented a 39% SD rate, as the best response, with rash and

diarrhea as the main toxicity events (Townsley et al. 2006).

This evidence of single agent erlotinib activity in metastatic

CRC patients led to the design of several trials in combination

with chemotherapy. The drug showed encouraging results

when used in combination with capecitabine and oxaliplatin in

previously treated disease in a phase II trial. In 32 patients pre-

treated with an irinotecan-containing regimen, 25% of the

patients experienced a partial response and 44% had stable

disease for at least 12 weeks (Meyerhardt et al. 2006).

In summary, the HER1/EGFR pathway proves to be a

valid target in GI cancers, especially in colorectal cancer.

Here we examined whether multi-targeting of HER family

receptors is a successful approach in colorectal cancer.

Given the diVerences in the mechanisms of action of these

drugs, we aimed to examine the antitumor activity of single

agent therapy with pertuzumab and in combination with

either erlotinib or irinotecan in human colon cancer cells in

vitro and in a murine xenograft model.

Materials and methods

Test agents and vehicles

Erlotinib hydrochloride was supplied as solution (Roche

Diagnostics GmbH, Penzberg, Germany) and stored at 4C.

A stock solution of pertuzumab (25 mg/ml) was obtained


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J Cancer Res Clin Oncol (2009) 135:13771386 1379

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from Roche Diagnostics GmbH (Penzberg, Germany). The

formulation vehicles were Captisol (sulfobutyl ether h-

cyclodextrin, sodium salt, 6% solution in water) and PBS

for erlotinib and pertuzumab. Dosing preparations of both

agents were prepared on the day of use. Irinotecan hydro-

chloride 3H2O (100 mg/5 ml) was obtained from PWzer

(New York, NY, USA).

Colorectal carcinoma cell lines and culture conditions

The cell lines WiDr, SW480, and SW620 were obtained

from the American Type Culture Collection (Rockville,

MD, USA). All other lines were kindly provided by M

Strauss/Berlin. Cells were maintained in Dulbeccos modi-

Wed Eagles Medium (DMEM) supplemented with antibiot-

ics and 10% fetal calf serum (FCS) (Gibco, Germany). The

cells were incubated at 37C in a humidiWed atmosphere

containing 10% CO2. Cells were passaged using trypsin/

EDTA (Gibco, Germany). On the day of tumor cell injec-

tion, cells were detached with trypsin/EDTA and washed in

culture medium. Cells were resuspended in DMEM, and

cell concentration and cell size were determined.

Preparation of proteins and western blot analysis

Colon cancer cell lines were tested for HER1/EGFR and

HER2 expression by western blots. Cells were lysed in NP-

40 lysis buVer (25 mM Tris HCl, pH 7.4, 0.5% NP-40,

100 mM NaCl, 1 mM EDTA), containing a protease inhibi-

tor cocktail (Roche, Germany) and 1 mM PMSF. Proteins

were resolved by sodium dodecyl sulfate-10% polyacryl-

amide gel electrophoresis (SDS-PAGE) and transferred to

Immobilon membranes (Millipore). HER1/EGFR, HER2

were detected using primary anti-HER1/EGFR and anti-

HER2 monoclonal antibodies from Santa Cruz Biotechnology

Inc. (Heidelberg, Germany) and Cell Signaling Technology

Inc. (Danvers, MA, USA). Additional goat anti-mouse and

anti-rabbit secondary antibodies labeled with Alexa Fluor

680 (700 nm, Molecular Probes, Invitrogen Corp) were

detected using Odyssey Infrared Imaging System (LI-COR

Biosciences, Lincoln, NE, USA).

Analysis of cell cycle distribution after pertuzumab

and EGF treatment

The eVect of pertuzumab on growth and cell cycle distribu-

tion was analyzed after stimulation with EGF ligand (R&D

Systems, Rsselsheim, Germany). Unstimulated cells and

stimulated cells incubated with BSA served as controls.

Cells were seeded onto 6-well plates and allowed to adhere

for 24 h. After serum starvation, cells were incubated alone

or with 10 g/ml pertuzumab for 30 min, then 100 ng/ml

EGF ligand was added. Cells were incubated with the

respective compound for 24 h and were harvested by tryp-

sinization. At the end of the treatment period, cells were

Wxed in 100% methanol for 30 min at 20C, centrifuged

for 10 min at 2000 rpm, resuspended in 0.1% Triton-X 100

in PBS containing propidium iodide (40 g/l; Sigma

Chemicals, Deisenhofen, Germany) and RNase (60 g/l;

Sigma Chemicals) and were incubated at 4C for a mini-

mum of 1 h. Subsequently, DNA content was measuredusing a Xow cytometer (Beckman Coulter, Krefeld, Germany)

and the cell cycle distribution was calculated using the

Phoenix Multicycle for Windows cell cycle analysis soft-

ware. A minimum of 10,000 events were measured for each

sample.

Animals

Female mice, Wve weeks old, were purchased from Harlan

Winkelmann (Borchen, Germany). Animals were housed in

suitable cages under speciWed pathogen-free conditions, in

rooms maintained at 23C and 50% humidity, with a 12-h

light/12-h dark cycle. The mice were quarantined during the

acclimatization period of at least a week. Standard food

(Altromin, Lage, Germany) and water were available

ad libitum. Regular health checks were done. Animal experi-

ments were performed in accordance with the Principles of

laboratory animal care (NIH publication no. 85-23, revised

1985) and according to local Committee Guidelines (GV-

Solas, Felasa, TierschG). The experimental study protocol

was reviewed and approved by the local government.

Growth inhibition studies in vivo

For establishment of xenograft tumors, mice were anesthe-

tized with ether (Otto Fischer GmbH & Co KG, Saarbrcken,

Germany). Two hundred-microliter suspensions of SW948,

DLD-1 or HCT116 tumor cells (Wnal concentration: 106

cells/200l) were transplanted s.c. into both Xanks of

NMRI nu/nu nude mice using a 1.0 ml tuberculin syringe

(needle 26 G 1 in., 0.45 25 mm; Becton Dickinson,

Drogheda, Ireland). Tumor-bearing mice were randomized

(n = 12 per group) when the mean tumor volume was

b(Prewett et al. 2002). At the end of the studies, all ani-

mals were sacriWced humanely by cervical dislocation

under anesthesia and tumors were evaluated histo-patho-

logically by light microscopy.

Randomized groups of tumor-bearing mice (n = 12) were

treated by oral gavage with vehicle (Captisol: 10 ml/kg


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per day) as control, or with erlotinib (25 mg/kg per day),

using 1 ml tuberculin syringes Wtted with feeding needles

with round tip and Luer lock hub (FST, Heidelberg,

Germany). The dose of erlotinib were based on previous

tolerability data. The i.p. dosing equipment was a 26 G 1

in., 0.45 25 mm, needle attached to a 1.0 ml tuberculin

syringe. The choice of pertuzumab dose and schedule was

based on previously reported data (Friess et al. 2005).Pertuzumab was administered i.p. at a loading dose of

12 mg protein/kg, followed by once weekly doses of 6 mg/

kg. In combination studies, groups of tumor-bearing mice

received erlotinib (25 mg/kg per day, p.o.) or irinotecan

(50100 mg/kg once a week, i.p.) and pertuzumab (6 mg/kg

once a week, i.p, with a twofold loading dose). Control ani-

mals were given vehicles, PBS i.p. for pertuzumab, NaCl

0.9% i.p. for irinotecan and captisol solution orally for erl-

otinib. Local animal welfare regulations require animals to

be terminated when tumors reach a certain size or show sur-

face ulceration. All animals were observed daily for clinical

signs of toxicity and weighed twice a week.

Statistical analysis

KruskalWallis test and Dunnett test were applied to com-

pare treatment groups with a level of 5% considered signiW-

cant using GraphPad Prism version 4 for Windows

(GraphPad Software, San Diego, CA, USA)

Histo-pathological analysis of explanted tumors

Samples of explanted tumors were Wxed in 4% buVered

formaldehyde (Kabe Labortechnik, Nmbrecht-Elsenroth,

Germany) and embedded in Paraplast (Sherwood Medical,

Norfolk, NE). From selected blocks, sections were cut, put

on coated slides (Superfrost plus, R. Langenbrinck, Teningen,

Germany), and dried for 12 h at 37C. The sections were

deparaYnized in xylene (J.T. Baker) and rehydrated

through graded concentrations of ethanol to distilled water.

For histopathologic examination, sections were stained

with H&E and examined by light microscopy (Leica

Microsystems, Wetzlar, Germany), by an experienced hist-

opathologist (IS.). Semiquantitative analysis of the tumor

slides using DISKUS software (Fa. Carl H. Hilgers,Knigswinter, Germany) included measurement of cell

necrosis and invasive growth.

Results

Various colon cancer cell lines express HER1/EGFR

and HER2

Nine diVerent colon cancer cell lines were tested for their

expression of HER1/EGFR (Fig. 1a) and HER2 (Fig. 1b)

by western blotting. The HER1/EGFR is a 170 kDa trans-

membrane receptor encoded by the human HER1/EGFR

gene and HER2 was detected as a protein of approximately

185 kDa. The cell lines SW480, HT29, DLD-1, LS174T,

HCT116 and Lovo showed the highest expression of the

receptors (Fig. 1a, b). Modest expression was seen in SW48

and SW948 colon cancer cells, and no expression was

detected in WIDR cell line. We detected robust HER1/

EGFR and HER2 overexpression in 67% of the nine colon

cancer cell lines.

EGF-induced cell cycle progression of colon cancer cells

is blocked by pertuzumab

We next tested the eVect of pertuzumab on cell cycle pro-

gression in colorectal cancer cells. First, we identiWed cell

Fig. 1 Western blots reveal

HER1/EGFR and HER2

expression of colon cancer cell

lines. Expression of HER1/

EGFR (a) and HER2 (b) in

colon cancer cells lines SW480,

HT 29, DLD-1, LS174T,

HCT116, SW948, Lovo, SW48

and WIDR was analyzed inlysates of conXuent monolayers

of cancer cell lines

170 kDa

HER1/EGFR150 kDa

250 kDa

185kDa

HER2

150 kDa

250 kDa

SW480

HT29

DLD-1

LS174T

HCT116

SW948

Lovo

SW48

Widr

50 KDa

Tubulin

50 KDa

Tubulin

A

B


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lines responsive to EGF ligand stimulation as detected by

cell cycle analysis. While cell lines SW480 (Fig. 2a),

HT115 (Fig. 2b) and SW948 (Fig. 2c) were clearly EGF

ligand responsive, cell lines Lovo, DLD-1, HT29 and

LS174T showed no alteration in cell cycle distribution

upon treatment with EGF. Consequently no inXuence of

pertuzumab on cell proliferation could be shown in these

cell lines. Subsequently after 24 h, we analyzed the eVect

on cell cycle distribution by Xow cytometry after treatment

with pertuzumab in combination with EGF ligand stimula-

tion or EGF stimulation alone as control (Fig. 2).

In EGF-dependent colon cancer cell lines SW480

(Fig. 2a), HT115 (Fig. 2b) and SW948 (Fig. 2c), EGF

induced an increase in S and G2 phase and a decrease in G1

phase as expected. Upon addition of pertuzumab, EGF-

induced cell cycle progression was partly reversed in

SW480, HT115 and SW948 approximating cell cycle dis-

tribution of control cells. Cell cycle progression in EGF

refractory HT29 cancer cells was not altered by pertuzumab

(Fig. 2c). The expression levels of HER1/EGFR and HER2

obviously did not predict the EGF response and the antipro-

liferative eVect of pertuzumab in vitro.

Pertuzumab inhibits growth of colon cancer xenograft

tumors in nude mice

EGF-induced cell cycle progression is blocked by pert-

uzumab most markedly in SW480 colon cancer cells, but

no stable growth of xenograft tumors was obtained with

this colon cancer cell line. The cell line SW948, on which

pertuzumab was eVective in vitro in reducing EGF-induced

cell cycle progression, was then successfully used to induce

Fig. 2 InXuence of pertuzumab

on cell cycle distribution. Cell

cycle distribution was deter-

mined by Xow cytometry 24 h

after treatment of colon cancer

cell lines SW480 (a), HT115 (b),

SW948 (c) and Lovo (d). The

graphsillustrate representative

results of cell cycle distribution

of untreated controls, and cells

treated either with EGF, or with

both EGF and pertuzumab

0

10

20

30

40

50

60

G1%

S%

G2%

SW480 Co SW480+EGF SW480+EGF

+pertuzumab

(%)

0

10

20

30

40

50

60

70

80

G1%

S%

G2%

HT115 Co HT115+EGF HT115+EGF

+pertuzumab

(%)

0

10

20

30

40

50

60

70

80

(%)

SW948 Co SW948+EGF SW948+EGF

0

10

20

30

40

50

60

70

G1%

S%

G2%

LovoCo Lovo+EGF Lovo+EGF

+pertuzumab

(%)

A B

C DG1%S%

G2%


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xenograft tumors in nude mice. After tumors were estab-

lished (approximately 510 days post injection into both

Xanks of NMRI nu/nu), mice were treated with either pert-

uzumab i.p. alone once a week, irinotecan i.p. alone once a

week, or with the combination of the two drugs, while PBS

served as control (n = 12/treatment arm with both Xanks of

mice injected). Pertuzumab antitumor activity on human

colon cancer xenograft tumors was examined in SW948(Fig. 3a) and HCT116 (Fig. 3b) in combination with irino-

tecan, and in DLD-1 xenograft tumors (Fig. 3c) in combi-

nation with erlotinib. Tumor volume was measured twice

weekly. While irinotecan strongly reduced tumor growth in

nude mice, the addition of pertuzumab to irinotecan in

SW948 (Fig. 3a) and HCT116 (Fig. 3b) xenograft tumors

did not enhance tumor growth reduction in vivo (Fig. 3a

and b). In these cell lines, pertuzumab monotherapy had a

small, non-signiWcant eVect on growth of xenograft tumors.

Subsequently, pertuzumab was combined with erlotinib

p.o. to further reduce HER1/EGFR signaling in colon can-

cer cells. Erlotinib was administered daily, while the mono-therapy of both agents or captisol served as control.

Pertuzumab showed minor antitumor activity in xenograft

tumors from DLD-1 cells (Fig. 3c), but highly signiWcantly

inhibited tumor growth when combined with the TKI erlotinib

Fig. 3 aGrowth inhibition of SW948 colon cancer xenografts in nude

mice. SW948 cells were established as xenograft tumors in femaleNMRI nu/nu mice. Tumors were allowed to establish growth after

implantation before initiation of treatment. Vehicle, CPT-11 (100 mg/

kg i.p. once weekly) and pertuzumab (12 mg/kg loading dose, fol-

lowed by 6 mg/kg i.p. once weekly), or CPT-11 with pertuzumab was

administered for the duration of the study. Tumor volume was deter-

mined twice weekly. Mean tumor volume (mm3) was plotted against

time (days). Symbolsmean tumor volume (mm3), barsSE (n = 12 mice

per group). b EVect of pertuzumab and/or irinotecan (CPT-11) on

growth of HCT116 human colon cancer cell xenograft tumors.

HCT116 cells were implanted s.c. (both Xanks) on anesthetized NMRI

nu/nu mice. Tumors were allowed to establish growth after implanta-

tion before initiation of treatment. Vehicle, CPT-11 (50 mg/kg i.p.

once weekly) and pertuzumab (12 mg/kg loading dose, followed by

6 mg/kg i.p. once weekly), or CPT-11 with pertuzumab was adminis-tered for the duration of the study. Mean tumor volume (mm 3) was

plotted against time (days). Symbolsmean tumor volume (mm3), bars

SE (n = 12 mice per group). cEVect of pertuzumab and erlotinib in

mono- and combination therapy on growth of DLD-1 human colon

cancer xenograft tumors. DLD-1 xenograft tumors were allowed to

establish growth after implantation before initiation of treatment.

Treatment of mice consisted of oral gavage of erlotinib or vehicle [erl-

otinib (25 mg/kg per day, orally), Captisol], pertuzumab i.p. (12 mg/kg

loading dose, following 6 mg/kg once weekly) or PBS i.p., or erlotinib

with pertuzumab for the duration of the study. Mean tumor volume

(mm3) was plotted against time (days). Symbolsmean tumor volume

(mm3), barsSE (n = 12 mice per group)

0

500

1000

1500

2000

2500

3000

1 4 8 11 15 18 22 25 29

1 5 8 12 15 22 2519

1 5 8 12 15 22 2519

PBS / NaCl

Pertuzumab

CPT-11

Pertuzumab/CPT-11

Meantumorvolume(mm

3)+SE

Meantumorvolume(mm

3)+SE

Meantumorvolume(m

m3)+SE

Treatment period (days)

0

100

200

300

400

500

600

700PBS / NaClPertuzumab

CPT-11

Pertuzumab/CPT-11


0

200

400

600

800

1000

1200

1400

1600Captisol/PBS

Pertuzumab/Captisol

Erlotinib/PBS

Pertuzumab/Erlotinib


A

C

B


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(mean tumor volume control: 1,065 mm3, erlotinib mono:

687 mm3, pertuzumab mono: 592 mm3, combination ther-

apy: 432 mm3, P < 0.001).

Histo-pathological examination of colon cancer xenograft

tumors

At the end of the 34 week treatment period, the xenograft

tumors were harvested and examined histologically after

H.E. staining showing intratumoral necrosis (Fig. 4). The

amount of necrosis was estimated upon light microscopy

using the DISKUS software. Necrosis was measured in

DLD-1 xenograft tumors after treatment with pertuzumab

alone and in combination with erlotinib. Untreated tumors

served as negative controls (Fig. 4). Histological examina-

tion after H&E staining of DLD-1 xenograft tumors

showed a slightly enhanced tumoral necrosis in pertuzumab

monotherapy, but not in combination therapy (Fig. 5a). The

amount of tumor necrosis in the treatment groups of pert-

uzumab (34.8%), erlotinib (33.8%) and in combination

(25.7%) was not increased compared with the control group

(37.8%). DiVerent results were obtained with colon cancer

cells HCT116 xenograft tumors (Fig. 5b), showing

enhanced tumor necrosis up to 57.4% in pertuzumab mono-

therapy, followed by necrosis up to 42% and 40.6% in the

CPT-11 mono and combination treatment arms. Here, the

controls showed the smallest amount of necrosis with

29.9%.

Discussion

Novel therapy strategies with targeting the ErbB network

may be achieved by inhibiting the tyrosine kinases cata-

lytic domain with small molecules (TKIs) or by inhibiting

the extracellular receptor domain with monoclonal antibod-

ies. In patients with colon cancer, epidermal growth factor

receptor (HER1/EGFR) is overexpressed or up-regulated in

6080% and HER2 overexpression has been previously

reported in up to 85% (Dursun et al. 2001; Mayer et al.

1993; Porebska et al. 2000; Ross and McKenna 2001). We

detected HER1/EGFR and HER2 overexpression in six of

nine colon cancer cell lines (Fig. 1). In a phase II trial in

metastatic colon cancer, HER2 was overexpressed in 8%

(11/138) of the patients tumor tissues. Partial response

(PR) was seen in Wve of seven evaluable patients treated

with irinotecan and trastuzumab. The study was closed due

to low accrual (Ramanathan et al. 2004). The partial

responses seen warranted the investigation of HER2 as a

potential therapy target in colon cancer.

Pertuzumab represents the Wrst HER2 dimerization

inhibitor tested in clinical trials of patients with various

solid tumors (Adams et al. 2006). Its antitumor activity is

not restricted to tumors with HER2 overexpression and is

diVerent from the therapeutic monoclonal antibody trast-

uzumab (Cobleigh et al. 1999; Franklin et al. 2004). There-

fore, our choice of cell lines was not limited by the HER2

expression status. Cell cycle analysis showed an EGF

induced cell cycle progression that was almost completely

abolished by pertuzumab in EGF-dependent colon cancer

cell lines SW480 (Fig. 2a), HT115 (Fig. 2b) and SW948

Fig. 4 Histo-pathological examination of colon cancer xenograft

tumors. H.E. staining showing intratumoral necrosis of xenograft

tumors after 34 weeks treatment. The amount of necrosis was deter-

mined using light microscopy

Fig. 5 Amount of necrosis in

colon cancer xenograft tumors

after therapy. The amount of

tumor necrosis per total tumor

volume (%) in the diVerent

treatment combinations was

measured upon light microscopy

in DLD-1 (a) and HCT116 (b)

xenograft tumors. The number

of examined tumors/group

varied between 19 and 24 0

10

20

30

40

50

60

0

10

20

30

40

50

60

70

80

necrosis(%)

necrosis(%)

treatment arm

Captisol

PBS

Pertuzumab

Captisol

Erlotinib

PBS

Erlotinib

Pertuzumab

PBS CPT-11 Pertuzu-

mab

Pertuzu-

mab

CPT-11treatment arm

A B


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(Fig. 2c). As expected, the antiproliferative eVect of pert-

uzumab in colon cancer cell lines in vitro was independent

from their HER1/EGFR and HER2 status.

Kuwada et al. demonstrated for the combination of

cetuximab and trastuzumab, an additive inhibition of cell

proliferation in EGF-responsive colon cancer cell lines

Caco-2 and HCA-7, compared to either antibody alone, but

showed no eVect on the cell proliferation of EGF refractorycancer cells DLD-1 and HT29 (Kuwada et al. 2004).

Our study of colon cancer cells in vitro revealed that

treatment with pertuzumab decreased the proliferation in

colon cancer cells. The examination of cell cycle analysis

by Xow cytometry was restricted to EGF responsive cancer

cells.

We further examined the antitumor activity of pert-

uzumab in vivo in colon cancer xenograft models based on

various colon cancer cell lines. The combination therapy

with pertuzumab and irinotecan was not superior to the

standard chemotherapy agent irinotecan. In xenograft

tumors from SW948 and HCT116 cell lines, pertuzumab

monotherapy had a modest eVect on tumor growth which

was statistically not signiWcant, probably because the

ranges of tumor sizes using these two cell lines were large

and the numbers of mice per treatment arm were too small

for pertuzumab monotherapy to reach signiWcance. While

irinotecan alone was very eVective in reducing tumor

growth, pertuzumab could not enhance the irinotecan eVect

on xenograft size.

Targeted therapies in clinical trials for colon cancer

patients failing standard therapy have gained at best a 10%

response rate, despite the fact that 80% of colon cancers

potentially use the HER network (Black et al. 2003; Norm-

anno et al. 2003). HER1/HER2 or HER2/HER3 heterodi-

mers may be responsible for the limited eYcacy and

targeting HER2 alone by monoclonal antibody might not be

suYcient for colon cancer treatment.

A previous study has shown that targeting HER2 with

pertuzumab failed to block GEO colon cancer cell prolifer-

ation when EGF is present in the medium (Jackson et al.

2004). More recently, a compensatory mechanism involv-

ing HER1/EGFR and HER2 for dimerization with HER3

was observed in GEO cells, when HER2 activation was

downregulated by a speciWc antibody or the selective HER2

TKI AG879 (Hu et al. 2005).

Because mabs and TKIs target the HER network at diVer-

ent sites, we decided to explore whether the combined admin-

istration of such compounds is superior to monotherapy. The

oral quinazoline erlotinib reversibly inhibits EGF receptor

tyrosine kinase and reduces intratumoral HER1/EGFR auto-

phosphorylation with no eVect on HER1/EGFR expression or

surface receptor density (Pollack et al. 1999).

In GEO, FET and HCT116 human colorectal cancer cell

lines, erlotinib diminished HER1/EGFR activation but did

not aVect total expression compared with controls. In con-

trast, HER2 activation was increased in all cell lines. The

inhibition of HER1/EGFR led to increased activation of

HER2. This result suggests a possible mechanism which

may rescue the cells after loss of anti-apoptotic signals

resulting from HER1/EGFR blockade. The inhibition of

multiple HER family members may yield stronger

responses than single receptor blockade (Learn et al. 2006).The convenient oral application of erlotinib and its

proven activity in phase II trial in colon cancer disease sug-

gested to test the combination approach of pertuzumab with

erlotinib (Meyerhardt et al. 2006). Clinical studies with erl-

otinib have shown that response to HER1/EGFR-targeted

therapy is not correlated with HER1/EGFR expression

(Prez-Soler et al. 2004).

We examined the eVect of pertuzumab and erlotinib in

mono- and combination therapy on growth of a well-estab-

lished DLD-1 xenograft tumor model. Pertuzumab alone

reduced tumor growth after 4 weeks of treatment in DLD-1

cells. Furthermore, when combined with erlotinib pert-

uzumab reduced tumor growth signiWcantly suggesting a

synergistic eVect of the two agents. The data presented here

suggest that the combination of diVerent classes of HER

inhibitors can augment the antitumor response over that

realized with a single HER inhibitor.

The combination of erlotinib and pertuzumab was shown

to be active against human non-small cell lung cancer

(NSCLC) (Calu-3, QG56) and breast cancer cell (KPL-4)

xenograft tumors, independently of their HER1/EGFR or

HER2 expression status (Friess et al. 2005).

Arpino et al. showed that combined treatment with geW-

tinib, trastuzumab, and pertuzumab blocked signals from

all HER homo- and heterodimers and inhibited growth of

HER2 overexpressing breast cancer xenografts of MCF7/

HER2-18 (HER2-transfected) or BT474 (HER2-ampliWed)

cells, signiWcantly better than single agents and dual combi-

nations. In the presence of the two HER2 antibodies trast-

uzumab and pertuzumab, geWtinib was needed for a

complete HER signaling blockade indicating that HER1/

EGFR signaling may be important in activating HER path-

ways, even at low levels of HER1/EGFR expression. After

blocking one HER pathway, tumors appeared to use an

alternative HER pathway (Arpino et al. 2007).

In the present study, the histo-pathological examination

of the HCT116 xenograft tumors revealed enhanced necro-

sis in pertuzumab monotherapy, which constituted approxi-

mately 55% of the area of the tumor sections, compared

with smaller amounts of necrosis in the other therapy and

control groups (Fig. 5a). These results are in line with pre-

vious preclinical Wndings. e.g., histo-pathological analysis

of ovarian carcinoma cell OVCA433 tumors from untreated

mice revealed approximately 20% necrotic areas in the

tumor sections. In contrast, 5060% of each of the tumor


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sections of mice treated with pertuzumab revealed necrosis,

histologic changes of apoptosis and Wbrosis involved

approximately 30% of the tumor area (Takai et al. 2005).

The histo-pathological examination of the DLD-1 xeno-

graft tumors in the present study did not indicate enhanced

tumor necrosis by combination therapy with pertuzumab

and erlotinib, in comparison to the monotherapy regimens.

Previous preclinical data using the EGF refractory cell lineDLD-1 and HT29 have shown no increased cancer cell

necrosis after 2 weeks of therapy with HER signaling

blocking antibodies trastuzumab and cetuximab (Kuwada

et al. 2004). However, Prewett el al. showed enhanced

tumor necrosis, tumor cell apoptosis and a reduction in

tumor vasculature in DLD-1 and HT29 xenograft tumors

treated with a combination of cetuximab and CPT-11

(Prewett et al. 2002). Large areas of necrosis in xenografts

may be indicative of ischemia due to the compromised

tumor vascularization following inhibition of HER1/EGFR

and HER2.

We showed in this preclinical study that pertuzumab

reduces the growth of colon cancer cells in vitro and in

vivo, in particular when combined with erlotinib. Both

drugs are known to have less severe side eVects than stan-

dard chemotherapeutic regimens. Pertuzumab treatment in

combination with irinotecan standard chemotherapy in the

murine xenograft model was not superior to monotherapy.

These preclinical data warrant further investigation of

simultaneous HER1/EGFR and HER2 dimerization inhibi-

tion for colorectal cancer treatment.

Acknowledgments The compounds erlotinib and pertuzumab were

supplied by Roche Diagnostics GmbH, Penzberg, Germany. This workwas supported by grants from the Forschungsfrderung der Ruhr-

Universitt (FoRUM), Germany.

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Antitumoral Activiti of HER2

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