Skip to main content
  • AACR Journals
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Focus on Radiation Oncology
      • Novel Combinations
      • Reviews
      • Editors' Picks
      • "Best of" Collection
  • First Disclosures
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Journals
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Molecular Cancer Therapeutics
Molecular Cancer Therapeutics
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Focus on Radiation Oncology
      • Novel Combinations
      • Reviews
      • Editors' Picks
      • "Best of" Collection
  • First Disclosures
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Research Articles

Effective Therapeutic Targeting of the Overexpressed HER-2 Receptor in a Highly Metastatic Orthotopic Model of Esophageal Carcinoma

Stephanie J. Gros, Nina Kurschat, Thorsten Dohrmann, Uta Reichelt, Ana-Maria Dancau, Kersten Peldschus, Gerhard Adam, Robert M. Hoffman, Jakob R. Izbicki and Jussuf T. Kaifi
Stephanie J. Gros
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nina Kurschat
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thorsten Dohrmann
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Uta Reichelt
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ana-Maria Dancau
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kersten Peldschus
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gerhard Adam
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert M. Hoffman
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jakob R. Izbicki
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jussuf T. Kaifi
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1158/1535-7163.MCT-10-0209 Published July 2010
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Abstract

This study aimed to determine the targeted efficacy of trastuzumab (Herceptin) on human epidermal growth factor receptor 2 (HER-2)-overexpressing metastatic esophageal cancer in an orthotopic mouse model. HER-2 overexpression and amplification of human esophageal primary and metastatic tumors were shown with HER-2–fluorescence in situ hybridization analysis and HER-2 immunostaining. Following orthotopic implantation with the HER-2–overexpressing OE19 human esophageal cancer cell line, mice were treated with trastuzumab. Sequential magnetic resonance imaging was used to monitor primary tumor and metastasis during treatment. After six weeks, a significant inhibition of primary tumor development was imaged in trastuzumab-treated animals in comparison with the control group. Trastuzumab treatment also led to a reduction of lymphatic metastasis. Thus, HER-2 targeted therapy with trastuzumab resulted in a significant primary tumor growth reduction as well as a decrease of lymph node metastases in the orthotopic model of metastatic esophageal carcinoma. The results of the present study suggest the clinical use of trastuzumab for HER-2–overexpressing esophageal cancer, which is a significant fraction of the patient population. Treatment of this highly treatment-resistant disease with trastuzumab in the adjuvant setting to prevent lymph node metastasis after primary tumor resection is suggested by the data in this report. Mol Cancer Ther; 9(7); 2037–45. ©2010 AACR.

Introduction

Human epidermal growth factor receptor 2 (HER-2) overexpression has been found in breast and other types of human cancer and has been developed as a therapeutic target (1–3). HER-2 gene amplification and protein overexpression are observed in about 20% of breast cancers (4) and are associated with a poor prognosis for these patients (5).

Antibody-based therapy with trastuzumab (Herceptin) is used clinically for targeting HER-2–positive breast cancer (6–8). Trastuzumab is most effective in HER-2–positive breast cancer patients when used as adjuvant therapy (9). There is also evidence for the possible efficacy of trastuzumab in HER-2–overexpressing cancers other than breast (10–12).

HER-2 overexpression was reported in esophageal cancer, with a tendency towards higher rates of positivity in adenocarcinoma (13–27) compared with squamous cell carcinomas (16, 19, 28–33).

A study of 110 esophageal adenocarcinoma patients has shown a strong concordance of HER-2 overexpression in primary and metastatic cancers with high-level HER-2 gene amplification. These data suggest esophageal cancer patients with HER-2–overexpressing primary tumors as candidates for trastuzumab therapy (34).

We had previously established an esophageal cancer surgical orthotopic implantation nude mouse model with organ and lymph node metastasis (35). In the present study, the surgical orthotopic implantation of the OE19 human esophageal cancer that overexpresses HER-2 was used. This nude mouse model exhibits the patterns of local and metastatic behavior occurring in clinical human esophageal carcinoma. With its high tumor take and metastatic frequency, it is a relevant model for investigating targeted therapies against primary tumor progression as well as metastatic spread. Small-animal magnetic resonance imaging (MRI) was used for real-time in vivo imaging of primary tumor and metastatic progression (35).

The aim of the present study was to evaluate the response to targeted trastuzumab therapy in this orthotopic esophageal adenocarcinoma model.

Materials and Methods

Cell line

The human esophageal carcinoma cell line OE19 was obtained from the European Collection of Cell Cultures, Health Protection Agency. The cells were cultured in RPMI 1640 medium (Biochrome KG) containing 10% fetal bovine serum (Linaris), penicillin/streptomycin (Biochrome KG), transferrin (Sigma-Aldrich), insulin (Sigma-Aldrich), basic fibroblast growth factor (Boehringer), and epidermal growth factor (Boehringer).

Fluorescence in situ hybridization

For proteolytic slide pretreatment, a commercial kit was utilized (Paraffin pretreatment reagent kit, Vysis). Spectrum Orange–labeled HER-2 probes were used together with Spectrum Green–labeled centromer 17 reference probes (PathVysiont, Vysis-Abbott). Before hybridization, sections were deparaffinized, air dried, dehydrated, and then denatured for 5 minutes at 74°C in 70% formamide-2× SSC solution. After overnight hybridization at 37°C in a humidified chamber, slides were washed and counterstained with 0.2 mmol/L 4′, 6-diamidino-2-phenylindole in an antifade solution. The mean numbers of HER-2 and centromer 17 signals were estimated for each tumor sample as previously described (36, 37). Our criterion for HER-2 gene amplification was a HER-2/centromer 17 signal ratio Z2. Low-level amplification ratio was defined as HER-2/centromer 17 at Z2 to 3. High-level amplification was defined as HER-2/centromer 17 ratio of Z3.

Immunohistochemistry of HER-2 expression

The HercepTest (DAKO) was used according to the manufacturer's protocol. Antigen retrieval of the deparaffinized tissue sections was done in a waterbath at 95°C to 99°C for 50 minutes followed by peroxidase blocking and incubation with the prediluted primary antibody. Cell line test slides provided by the manufacturer were used as positive and negative controls. Immunostaining was scored by one pathologist (U.R.), following a four-step scale (0, 1þ, 2þ, 3þ) according to the manufacturer's directions.

RNA extraction and HER-2 amplification

OE19 cancer cells were harvested after trypsinization and washed three times, and 5 × 106 cells were used for RNA extraction using the PARIS Kit (Ambion). This was repeated for MDA-MB-231 and SKBr-3 cells as positive controls. cDNA was obtained with an Invitrogen reverse transcriptase-PCR kit. Amplification of HER-2 DNA was done with a Thermocycler (Biometra) with Taq-Polymerase (Roche) and HER-2–specific primers (GAGCCGCGAGCACCCAAGT, TCCATTGTCTAGCACGGCCA) as well as GAPDH primers as controls (ACCACAGTCCATGCCATCAC, TCCACCACCCTGTTGCTGTA). PCR products were run on a 2% agarose gel. Imaging was done with a Biodoc II (Biometra). For size reference, a 100 bp DNA ladder (New England Biolabs Inc.) was used.

Cell proliferation assay

The effect of trastuzumab on tumor cell growth was examined with a nonradioactive cell proliferation assay (MTT, Promega). OE19 cells were cultured in 96-well plates. Each well contained 10,000 cells in 100 μL RPMI 1640 medium (Linaris) with 10% FCS (Linaris). After overnight incubation, the medium was removed and replaced with RPMI 1640 plus 10% FCS for control and 20 μg/mL trastuzumab (Roche) in RPMI 1640 plus 10% FCS. One plate was used for determination of the starting concentration. MTT reagent (20 μL) was added. The extinction was measured in an enzyme-linked immunosorbent assay reader (Microplate Reader, Dynatech MR500) after 2 hours of incubation at 37°C. The remaining plates were incubated for 72 hours and the extinction was measured as mentioned above.

Orthotopic esophageal carcinoma mouse model

NMRI/nu/nu (U.S. Naval Medical Research Institute) nude mice were obtained from Charles River Deutschland at 10 weeks of age and housed in the animal facility of the University Medical Center Hamburg-Eppendorf. All animal procedures were done in accordance with a protocol approved by the Behörde für Wissenschaft und Gesundheit (Freie und Hansestadt Hamburg, Germany). OE19 cells (5 × 106), in a 200 μL suspension, were s.c. injected into the flanks of nude mice with a 1 mL syringe (BD Plastipak, Becton Dickinson S.A.) with a 27-gauge hypodermic needle (Sterican) within 40 minutes of harvesting. The mice were weighed and examined for tumor development every other day. When tumor growth reached 5 to 7 mm, tumors were excised. Esophageal tumor fragments (1 mm3), derived from the s.c. tumors, were orthotopically implanted to the abdominal esophagus (38). Mice were anaesthetized with ketamine hydrochloride (Graeub)/xylazine hydrochloride (Bayer) mixture (12 mg/mL) and i.p. injected at 10 ml/kg body weight. A 0.8-cm transverse incision in the skin of the epigastric abdomen was made. The abdominal muscles and peritoneum were separated by sharp dissection, and the abdomen was opened. The great curvature of the stomach was held by forceps, and the liver was raised to expose the abdominal esophagus. A lesion of the esophageal serosa was made with sharp forceps. Four tumor fragments were sutured to this lesion with 8.0 prolene sutures (Ethicon). The incision of the abdominal wall was closed using 6.0 vicryl sutures (Ethicon). All procedures of the operation, as described above, were done under an operating dissecting microscope (Carl Zeiss). Postoperative analgesia was achieved by novamine sulfone (1 mg/mL) in drinking water. The mice were weighed and examined for tumor development every other day.

Therapy with trastuzumab (Herceptin)

After primary tumor growth was determined by MRI on day 14, the mice were randomized into two groups. Group 1 was treated biweekly with an i.p. injection of 20 mg/kg body weight trastuzumab (Roche) in a volume of 100 μL. Group 2 was given sham injections with 100 μL PBS and was used as a control group.

Magnetic resonance imaging

Whole-body MRI of tumor-bearing mice was done to assess the localization and size of tumors under ketamine hydrochloride/xylacine hydrochloride anesthesia as described above. High-resolution magnetic resonance data sets were acquired on a clinical 3 Tesla whole-body magnetic resonance scanner (Intera, Philips Medical Systems) equipped with a standard gradient system (gradient strength = 40 mT/m). A dedicated small-animal solenoid receiver coil (Philips Research) with an inner diameter of 40 mm and an integrated heating system to regulate the body temperature of mice during magnetic resonance scans was used for signal exploitation. The magnetic resonance protocol consisted of a short survey scan and three T2-weighted two-dimensional turbo spin-echo sequences in coronal, sagittal, and axial orientations. Imaging parameters were as follows: repetition time, 2,674 ms; echo time, 90 ms; echo train length, 10; number of excitations, 2. The acquisition matrix and field of view (FOV) were adopted to display the animals in each orientation with an in-plane resolution of 200 × 200 μm. For coronal and sagittal images, a matrix of 400 × 200 pixels and a FOV of 80 × 40 mm were used. Axial images were acquired with a matrix of 160 × 128 pixels and a FOV of 32 × 25.6 mm. Slice parameters were equal for all three sequences with a thickness of 800 μm and 14 slices. The total scan time per animal was 10 minutes and 42 seconds. Image analysis was done on a conventional clinical workstation. Suspicious lesions were considered to be tumors if they appeared hyperintense on the T2-weighted images and had a diameter ≥1 mm.

Examination of esophageal carcinoma at necropsy: primary tumor and metastases

After 6 weeks of treatment, the mice were sacrificed and autopsied. The orthotopic primary tumor as well as lung, liver, and axillary, mediastinal, pancreatic, renal, mesenteric, and inguinal lymph nodes were dissected. Fragments of metastatic liver, lung, and lymph node that were macroscopically visible were placed in Hank's solution for growth of metastatic cell cultures. All other tissues were preserved in formalin and cryopreserved.

Results

HER-2 expression in the OE19 esophageal carcinoma in vitro and in the orthotopic model

In comparison with MD-MB-231 and SKBr-3 breast cancer cells, esophageal carcinoma OE19 cells showed high levels of HER-2 mRNA (Fig. 1A) and thus were chosen for trastuzumab targeted treatment in the orthotopic model. One hundred percent of the mice developed primary tumor growth after implantation of OE19 cell fragments to the abdominal esophagus (Table 1). As we had also previously reported with the human 1590-GFP esophageal carcinoma cell line (35), the primary tumor remained attached to the abdominal esophagus, infiltrated the serosa, but did not cause intraluminal stenosis (Fig. 1B). Immunohistochemical staining of tumor cells showed an intensely positive signal for HER-2 (Fig. 1B). The primary tumor resulted in spontaneous metastases to the liver, lung, and lymph nodes. Macroscopic metastases were recorded by open photography (Fig. 1C).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

A, HER-2 mRNA expression in the human esophageal carcinoma cell line OE19 is elevated. B, HER-2–positive primary tumor (arrows) is located at the abdominal esophagus (star) infiltrating the esophageal serosa. C, extensive liver metastases (arrows) can often be identified macroscopically at autopsy. These results are from typical experiments.

View this table:
  • View inline
  • View popup
Table 1.

Efficacy of trastuzumab on primary and metastatic esophageal cancer

Inhibition of in vitro OE19 cell proliferation by trastuzumab

Trastuzumab inhibited the proliferation of OE19 cells in vitro. A significant difference (P = 0.045) of relative cell proliferation was observed in cell cultures treated with trastuzumab (Fig. 2A).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

A significant inhibition of cell proliferation as well as primary tumor growth reduction was seen in vitro (A) as well as in vivo (B) by trastuzumab. HER-2 targeted therapy also led to inhibition of lymph node metastases (C).

Trastuzumab targeting of primary tumor growth

Two weeks after surgical orthotopic implantation of OE19, primary tumor growth was confirmed by MRI. Treatment started at this time. In the treatment group, eight mice were treated with i.p. trastuzumab twice a week. In the control group, eight mice were treated with i.p. PBS of the same volume at the same times as the treatment group. One mouse in the control group died on day 11 after the beginning of treatment due to causes unrelated to tumor growth or therapy and was thus excluded from further analysis. Sequential MRI was done throughout the experiment. At the termination date, after 6 weeks of treatment, the mice were sacrificed.

One hundred percent of the mice in the treated and the untreated groups had primary tumor growth on the abdominal esophagus. Primary tumors were dissected and weighed. Tumor weights ranged between 0.7 g and 5.4 g with a mean of 2.36 g in the control group and between 0.3 g and 1.8 g with a mean of 1.09 g in the trastuzumab-treated group (Table 1A and B), showing a significant difference (P = 0.044) between the groups (Fig. 2B). Trastuzumab targeting resulted in significantly reduced primary tumor size. No adverse effects of trastuzumab therapy were observed.

Trastuzumab targeting of metastasis

Primary tumor, parenchymal organs, and lymph nodes were dissected at necropsy and prepared for further analysis. Upon histologic examination, metastases were seen in 71% of animals in the control group, whereas 50% of animals in the trastuzumab-treated group showed metastases (Table 1A and B). Considering only lymphatic metastasis, 71% of the animals in the control group had lymph node metastases whereas 37.5% of animals in the trastuzumab-treated group had positive lymph nodes (Fig. 2C). Trastuzumab targeting thus also leads to a reduction of lymphatic spread.

Primary tumors and liver, lung, and lymph node metastases showed amplification of HER-2 in fluorescence in situ hybridization analysis. An example is given for primary tumor amplification in Fig. 3A. All of the primary tumors and metastases in the treated and the untreated groups, regardless of the metastatic organ (liver, lung, lymph node), expressed high levels of HER-2 as seen by immunostaining (Fig. 3B).

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

HER-2 expression in esophageal cancer cell line OE19. Orthotopic primary tumor, and liver, lung, and lymph node metastases. OE19 cell line showed high level HER-2 amplification (A). Orthotopic primary tumor, and metastases to the liver, lung, and lymph node showed high-level HER-2 overexpression (B) throughout. These results are from typical experiments. FISH, fluorescence in situ hybridization; PT, primary tumor; LN, lymph node; H.E., hematoxylin and eosin staining.

In vivo tumor imaging

Sequential in vivo MRI of control and treated animals was done every two weeks after orthotopic implantation. The final imaging was done on the day of sacrifice. Primary tumor growth could be confirmed in all cases two weeks after orthotopic implantation. Before the start of treatment, primary tumor imaging showed comparable tumor sizes in untreated and treated groups (Fig. 4A and C) two weeks after orthotopic implantation. Over the time course of the treatment, slower progression of tumor size could be observed in sequential imaging in the trastuzumab-treated group compared to the untreated control, leading to significant imageable tumor size difference at the time of termination of the experiment (Fig. 4B and D).

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

Serial MRI confirmed inhibition of primary tumor progression in trastuzumab targeting at the beginning and end point of treatment in comparison with the untreated group. Arrows, primary tumors.

Discussion

Orthotopic models are essential in the understanding of primary tumor progression in vivo and the biology of metastatic spread (38, 39). The esophageal carcinoma cell line OE19 overexpresses HER-2 and thus is ideal for trastuzumab targeting. The orthotopic model showed overexpression of HER-2 in the primary tumor, as well as liver, lung, and lymph node metastases.

The tumor take rate in this OE19 orthotopic tumor model was 100%. Metastatic frequency in this study was 71% in the untreated group. The high primary tumor take and metastatic frequency allowed evaluation of trastuzumab targeting of the primary tumor as well as metastasis.

Orthotopic models have previously been successfully used for evaluation of trastuzumab therapy for other tumor types (40–42). The efficacy of trastuzumab on HER-2–positive breast cancer has been shown in vitro and in orthotopic models as well as clinically in breast cancer patients (6–9).

There is also evidence for a possible response of HER-2–positive cancers other than breast to trastuzumab (10–12). Several studies have suggested that HER-2 amplification/overexpression may be relevant for these tumor types, but the potential benefit of trastuzumab in tumors other than breast is largely unknown.

Inhibition of metastasis by trastuzumab has been described in orthotopic human pancreatic cancer xenografts with low-level HER-2/neu expression (42). Incidence of metastases was reduced, especially in the liver.

There is evidence that HER-2 overexpression is involved in progression from dysplasia in patients with Barrett's esophagus to adenocarcinoma (43), and targeted treatment with trastuzumab has been attempted (44).

HER-2 overexpression was reported in up to 83% of esophageal cancer, with a tendency towards higher rates of positivity in adenocarcinoma (10–83%; refs. 13–27) compared with squamous cell carcinomas (up to 56%; refs. 16, 19, 28–33). A similar variability was observed in amplification analyses.

One phase I-II trial was conducted to assess the effects of trastuzumab in the treatment of patients with esophageal adenocarcinoma overexpressing HER-2 in combination with paclitaxel, cisplatin, and radiation (45). This study was limited by a small number of patients. No increase in toxicity due to trastuzumab was observed.

In an analysis of our patient population, we previously reported a HER-2 amplification of 15% in esophageal adenocarcinoma patients (16 of 110; ref. 34). We found a strong concordance of the HER-2 status in primary and metastatic esophageal cancer with high-level HER-2 gene amplification. Such patients will be candidates for trastuzumab treatment, preferably in the adjuvant setting, because our results show inhibition of metastasis, especially lymph node metastasis (34).

Our results show a significant reduction of esophageal adenocarcinoma tumor cell proliferation by trastuzumab. In vitro a significant reduction of cell proliferation by trastuzumab (P = 0.045) was observed. In vivo there was a significant reduction of primary tumor growth (P = 0.044) in the orthotopic model in the trastuzumab-treated group in comparison with the untreated group. Metastasis was also inhibited. There was no adverse effect to trastuzumab therapy observed in vivo.

It has already been shown that trastuzumab blocks cancer cells in the cell cycle (46–49) and induces apoptosis. Furthermore sensitivity of OE19 cells to trastuzumab has previously been shown (50).

In the literature, HER-2 positivity rates in esophageal adenocarcinoma are up to 83% (13–27). Therefore, targeted therapy with trastuzumab in HER-2–positive esophageal carcinoma seems to be an option in the individualized treatment.

There is a discussion on the risk of central nervous system metastases in patients treated with or without trastuzumab. Trastuzumab, which is a large monoclonal antibody, does not penetrate the blood-brain barrier and, thus, may allow the brain to become a sanctuary site for micrometastases. The results of a study by Lai and colleagues did not support an association between trastuzumab therapy and an increased risk of central nervous system metastases (51). However, several other studies report a higher risk in treated patients (52–55).

A Chinese study on the correlation between HER-2 gene amplification and lymphangiogenesis and their prognostic significance in human breast cancer suggests that by upregulating vascular endothelial growth factor C expression, HER-2 overexpression induces lymphangiogenesis and thus promotes metastasic spread (56).

The efficacy of trastuzumab on tumor angiogenesis will be determined in future experiments.

In our highly metastatic orthotopic model of esophageal carcinoma, the overall metastatic rate in vivo was reduced from 71% in the untreated to 50% in the trastuzumab-treated group. Lymph node metastases were seen in 71% of untreated animals, whereas the metastatic rate was reduced to 37.5% in the trastuzumab-treated group. These results show a strong effect of trastuzumab therapy on the primary tumor as well as on lymph node metastases. The results of the present study suggest the clinical use of trastuzumab for HER-2–overexpressing esophageal cancer, which is a significant fraction of the patient population with this disease. In particular, treatment with trastuzumab could be used in the adjuvant setting to prevent lymph node metastasis after primary tumor resection.

Sequential in vivo small-animal MRI allows noninvasive imaging of esophageal carcinoma progression over the course of the experiment. After confirmation of primary tumor growth (tumor take rate of 100%), treatment can be started and the effect of treatment monitored over time. Responsiveness to therapeutic treatment can be evaluated in real time with this highly sensitive imaging technique.

In conclusion, HER-2 targeted therapy with trastuzumab (Herceptin) shows a significant primary tumor growth reduction as well as a reduction of lymph node metastases in an orthotopic model of metastatic esophageal carcinoma. These preclinical results suggest a role for HER-2 targeted antibody-based treatment of HER-2–overexpressing esophageal carcinoma. The results suggest, in particular, trastuzumab treatment in the adjuvant setting to prevent lymph node metastasis after primary tumor resection.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Antje Heinecke for expert technical assistance.

Grant Support: Deutsche Forschungsgemeinschaft grant GR 3484/1-1.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

    • Received March 2, 2010.
    • Revision received May 10, 2010.
    • Accepted May 10, 2010.
    • ©2010 American Association for Cancer Research.

    References

    1. ↵
      1. Hynes NE,
      2. Lane HA
      . ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005;5:341–54.
      OpenUrlCrossRefPubMed
      1. Normanno N,
      2. Bianco C,
      3. Strizzi L,
      4. et al
      . The ErbB receptors and their ligands in cancer: an overview. Curr Drug Targets 2005;6:243–57.
      OpenUrlCrossRefPubMed
    2. ↵
      1. Rabindran SK
      . Antitumor activity of HER-2 inhibitors. Cancer Lett 2005;227:9–23.
      OpenUrlCrossRefPubMed
    3. ↵
      1. Zhang D,
      2. Salto-Tellez M,
      3. Do E,
      4. Putti TC,
      5. Koay ES
      . Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays. Hum Pathol 2003;34:362–8.
      OpenUrlCrossRefPubMed
    4. ↵
      1. Slamon DJ,
      2. Clark GM,
      3. Wong SG,
      4. Levin WJ,
      5. Ullrich A,
      6. McGuire WL
      . Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–82.
      OpenUrlAbstract/FREE Full Text
    5. ↵
      1. Baselga J
      . The EGFR as a target for anticancer therapy-focus on cetuximab. Eur J Cancer 2001;37 Suppl 4:S16–22.
      OpenUrlPubMed
      1. Leyland-Jones B
      . Trastuzumab: hopes and realities. Lancet Oncol 2002;3:137–44.
      OpenUrlCrossRefPubMed
    6. ↵
      1. Tripathy D,
      2. Slamon DJ,
      3. Cobleigh M,
      4. et al
      . Safety of treatment of metastatic breast cancer with trastuzumab beyond disease progression. J Clin Oncol 2004;22:1063–70.
      OpenUrlAbstract/FREE Full Text
    7. ↵
      1. Tuma RS
      . Trastuzumab trials steal show at ASCO meeting. J Natl Cancer Inst 2005;97:870–1.
      OpenUrlFREE Full Text
    8. ↵
      1. Kollmannsberger C,
      2. Pressler H,
      3. Mayer F,
      4. Kanz L,
      5. Bokemeyer C
      . Cisplatin-refractory, HER-2/neu-expressing germ-cell cancer: induction of remission by the monoclonal antibody Trastuzumab. Ann Oncol 1999;10:1393–4.
      OpenUrlFREE Full Text
      1. Langer CJ,
      2. Stephenson P,
      3. Thor A,
      4. Vangel M,
      5. Johnson DH
      . Trastuzumab in the treatment of advanced non-small-cell lung cancer: is there a role? Focus on Eastern Cooperative Oncology Group study 2598. J Clin Oncol 2004;22:1180–7.
      OpenUrlAbstract/FREE Full Text
    9. ↵
      1. Locati LD,
      2. Rinaldi G,
      3. Bossi P,
      4. et al
      . Herceptin plus chemotherapy in relapsed and/or metastatic salivary gland cancer. Oral Oncol 2005;41:97–8.
      OpenUrlCrossRefPubMed
    10. ↵
      1. al-Kasspooles M,
      2. Moore JH,
      3. Orringer MB,
      4. Beer DG
      . Amplification and over-expression of the EGFR and erbB-2 genes in human esophageal adenocarcinomas. Int J Cancer 1993;54:213–9.
      OpenUrlPubMed
      1. Duhaylongsod FG,
      2. Gottfried MR,
      3. Iglehart JD,
      4. Vaughn AL,
      5. Wolfe WG
      . The significance of c-erb B-2 and p53 immunoreactivity in patients with adenocarcinoma of the esophagus. Ann Surg 1995;221:677–83.
      OpenUrlPubMed
      1. Flejou JF,
      2. Paraf F,
      3. Muzeau F,
      4. et al
      . Expression of c-erbB-2 oncogene product in Barrett's adenocarcinoma: pathological and prognostic correlations. J Clin Pathol 1994;47:23–6.
      OpenUrlAbstract/FREE Full Text
    11. ↵
      1. Friess H,
      2. Fukuda A,
      3. Tang WH,
      4. et al
      . Concomitant analysis of the epidermal growth factor receptor family in esophageal cancer: overexpression of epidermal growth factor receptor mRNA but not of c-erbB-2 and c-erbB-3. World J Surg 1999;23:1010–8.
      OpenUrlCrossRefPubMed
      1. Geddert H,
      2. Zeriouh M,
      3. Wolter M,
      4. Heise JW,
      5. Gabbert HE,
      6. Sarbia M
      . Gene amplification and protein overexpression of c-erb-b2 in Barrett carcinoma and its precursor lesions. Am J Clin Pathol 2002;118:60–6.
      OpenUrlAbstract/FREE Full Text
      1. Hardwick RH,
      2. Shepherd NA,
      3. Moorghen M,
      4. Newcomb PV,
      5. Alderson D
      . c-erbB-2 overexpression in the dysplasia/carcinoma sequence of Barrett's oesophagus. J Clin Pathol 1995;48:129–32.
      OpenUrlAbstract/FREE Full Text
    12. ↵
      1. Hardwick RH,
      2. Barham CP,
      3. Ozua P,
      4. et al
      . Immunohistochemical detection of p53 and c-erbB-2 in oesophageal carcinoma; no correlation with prognosis. Eur J Surg Oncol 1997;23:30–5.
      OpenUrlCrossRefPubMed
      1. Jankowski J,
      2. Coghill G,
      3. Hopwood D,
      4. Wormsley KG
      . Oncogenes and onco-suppressor gene in adenocarcinoma of the oesophagus. Gut 1992;33:1033–8.
      OpenUrlAbstract/FREE Full Text
      1. Kim R,
      2. Clarke MR,
      3. Melhem MF,
      4. et al
      . Expression of p53, PCNA, C-erbB-2 in Barrett's metaplasia and adenocarcinoma. Dig Dis Sci 1997;42:2453–62.
      OpenUrlCrossRefPubMed
      1. Nakamura T,
      2. Nekarda H,
      3. Hoelscher AH,
      4. et al
      . Prognostic value of DNA ploidy and c-erbB-2 oncoprotein overexpression in adenocarcinoma of Barrett's esophagus. Cancer 1994;73:1785–94.
      OpenUrlCrossRefPubMed
      1. Safran H,
      2. Dipetrillo T,
      3. Nadeem A,
      4. et al
      . Trastuzumab, paclitaxel, cisplatin, and radiation for adenocarcinoma of the esophagus: a phase I study. Cancer Invest 2004;22:670–7.
      OpenUrlCrossRefPubMed
      1. Sauter ER,
      2. Keller SM,
      3. Erner S,
      4. Goldberg M
      . HER-2/neu: a differentiation marker in adenocarcinoma of the esophagus. Cancer Lett 1993;75:41–4.
      OpenUrlCrossRefPubMed
      1. Trudgill NJ,
      2. Suvarna SK,
      3. Royds JA,
      4. Riley SA
      . Cell cycle regulation in patients with intestinal metaplasia at the gastro-oesophageal junction. Mol Pathol 2003;56:313–7.
      OpenUrlAbstract/FREE Full Text
      1. Walch A,
      2. Bink K,
      3. Gais P,
      4. et al
      . Evaluation of c-erbB-2 overexpression and Her-2/neu gene copy number heterogeneity in Barrett's adenocarcinoma. Anal Cell Pathol 2000;20:25–32.
      OpenUrlPubMed
    13. ↵
      1. Walch A,
      2. Specht K,
      3. Bink K,
      4. et al
      . Her-2/neu gene amplification, elevated mRNA expression, and protein overexpression in the metaplasia-dysplasia-adenocarcinoma sequence of Barrett's esophagus. Lab Invest 2001;81:791–801.
      OpenUrlPubMed
    14. ↵
      1. Akamatsu M,
      2. Matsumoto T,
      3. Oka K,
      4. et al
      . c-erbB-2 oncoprotein expression related to chemoradioresistance in esophageal squamous cell carcinoma. Int J Radiat Oncol Biol Phys 2003;57:1323–7.
      OpenUrlCrossRefPubMed
      1. Lam KY,
      2. Tin L,
      3. Ma L
      . C-erbB-2 protein expression in oesophageal squamous epithelium from oesophageal squamous cell carcinomas, with special reference to histological grade of carcinoma and pre-invasive lesions. Eur J Surg Oncol 1998;24:431–5.
      OpenUrlCrossRefPubMed
      1. Mimura K,
      2. Kono K,
      3. Hanawa M,
      4. et al
      . Frequencies of HER-2/neu expression and gene amplification in patients with oesophageal squamous cell carcinoma. Br J Cancer 2005;92:1253–60.
      OpenUrlCrossRefPubMed
      1. Suo Z,
      2. Holm R,
      3. Nesland JM
      . Squamous cell carcinomas. An immunohistochemical study of cytokeratins and involucrin in primary and metastatic tumours. Histopathology 1993;23:45–54.
      OpenUrlCrossRefPubMed
      1. Suo Z,
      2. Su W,
      3. Holm R,
      4. Nesland JM
      . Lack of expression of c-erbB-2 oncoprotein in human esophageal squamous cell carcinomas. Anticancer Res 1995;15:2797–8.
      OpenUrlPubMed
    15. ↵
      1. Suwanagool P,
      2. Parichatikanond P,
      3. Maeda S
      . Expression of c-erbB-2 oncoprotein in primary human tumors: an immunohistochemistry study. Asian Pac J Allergy Immunol 1993;11:119–22.
      OpenUrlPubMed
    16. ↵
      1. Reichelt U,
      2. Duesedau P,
      3. Tsourlakis MC,
      4. et al
      . Frequent homogeneous HER-2 amplification in primary and metastatic adenocarcinoma of the esophagus. Mod Pathol 2007;20:120–9.
      OpenUrlCrossRefPubMed
    17. ↵
      1. Gros SJ,
      2. Dohrmann T,
      3. Peldschus K,
      4. et al
      . Complementary use of fluorescence and magnetic resonance imaging of metastatic esophageal cancer in a novel orthotopic mouse model. Int J Cancer 2010;126:2671–81.
      OpenUrlPubMed
    18. ↵
      1. Bubendorf L,
      2. Kolmer M,
      3. Kononen J,
      4. et al
      . Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays. J Natl Cancer Inst 1999;91:1758–64.
      OpenUrlAbstract/FREE Full Text
    19. ↵
      1. Schraml P,
      2. Kononen J,
      3. Bubendorf L,
      4. et al
      . Tissue microarrays for gene amplification surveys in many different tumor types. Clin Cancer Res 1999;5:1966–75.
      OpenUrlAbstract/FREE Full Text
    20. ↵
      1. Hoffman RM
      . Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 1999;17:343–59.
      OpenUrlCrossRefPubMed
    21. ↵
      1. Hoffman RM
      . Orthotopic metastatic (MetaMouse) models for discovery and development of novel chemotherapy. Methods Mol Med 2005;111:297–322.
      OpenUrlPubMed
    22. ↵
      1. Francia G,
      2. Man S,
      3. Lee CJ,
      4. et al
      . Comparative impact of trastuzumab and cyclophosphamide on HER-2-positive human breast cancer xenografts. Clin Cancer Res 2009;15:6358–66.
      OpenUrlAbstract/FREE Full Text
      1. Gee MS,
      2. Upadhyay R,
      3. Bergquist H,
      4. et al
      . Human breast cancer tumor models: molecular imaging of drug susceptibility and dosing during HER2/neu-targeted therapy. Radiology 2008;248:925–35.
      OpenUrlCrossRefPubMed
    23. ↵
      1. Pratesi G,
      2. Petrangolini G,
      3. Tortoreto M,
      4. et al
      . Antitumor efficacy of trastuzumab in nude mice orthotopically xenografted with human pancreatic tumor cells expressing low levels of HER-2/neu. J Immunother 2008;31:537–44.
      OpenUrlCrossRefPubMed
    24. ↵
      1. Rossi E,
      2. Grisanti S,
      3. Villanacci V,
      4. et al
      . HER-2 overexpression/amplification in Barrett's oesophagus predicts early transition from dysplasia to adenocarcinoma: a clinico-pathologic study. J Cell Mol Med 2009;13:3826–33.
      OpenUrlCrossRefPubMed
    25. ↵
      1. Villanacci V,
      2. Rossi E,
      3. Grisanti S,
      4. et al
      . Targeted therapy with trastuzumab in dysplasia and adenocarcinoma arising in Barrett's esophagus: a translational approach. Minerva Gastroenterol Dietol 2008;54:347–53.
      OpenUrlPubMed
    26. ↵
      1. Safran H,
      2. Dipetrillo T,
      3. Akerman P,
      4. et al
      . Phase I/II study of trastuzumab, paclitaxel, cisplatin and radiation for locally advanced, HER-2 overexpressing, esophageal adenocarcinoma. Int J Radiat Oncol Biol Phys 2007;67:405–9.
      OpenUrlPubMed
    27. ↵
      1. Peng D,
      2. Fan Z,
      3. Lu Y,
      4. DeBlasio T,
      5. Scher H,
      6. Mendelsohn J
      . Anti-epidermal growth factor receptor monoclonal antibody 225 up-regulates p27KIP1 and induces G1 arrest in prostatic cancer cell line DU145. Cancer Res 1996;56:3666–9.
      OpenUrlAbstract/FREE Full Text
      1. Kawaguchi Y,
      2. Kono K,
      3. Mimura K,
      4. et al
      . Targeting EGFR and HER-2 with cetuximab- and trastuzumab-mediated immunotherapy in oesophageal squamous cell carcinoma. Br J Cancer 2007;97:494–501.
      OpenUrlCrossRefPubMed
      1. Liu B,
      2. Fang M,
      3. Lu Y,
      4. Mendelsohn J,
      5. Fan Z
      . Fibroblast growth factor and insulin-like growth factor differentially modulate the apoptosis and G1 arrest induced by anti-epidermal growth factor receptor monoclonal antibody. Oncogene 2001;20:1913–22.
      OpenUrlCrossRefPubMed
    28. ↵
      1. Mimura K,
      2. Kono K,
      3. Hanawa M,
      4. et al
      . Trastuzumab-mediated antibody-dependent cellular cytotoxicity against esophageal squamous cell carcinoma. Clin Cancer Res 2005;11:4898–904.
      OpenUrlAbstract/FREE Full Text
    29. ↵
      1. Dahlberg PS,
      2. Jacobson BA,
      3. Dahal G,
      4. et al
      . ERBB2 amplifications in esophageal adenocarcinoma. Ann Thorac Surg 2004;78:1790–800.
      OpenUrlCrossRefPubMed
    30. ↵
      1. Lai R,
      2. Dang CT,
      3. Malkin MG,
      4. Abrey LE
      . The risk of central nervous system metastases after trastuzumab therapy in patients with breast carcinoma. Cancer 2004;101:810–6.
      OpenUrlCrossRefPubMed
    31. ↵
      1. Kallioniemi OP,
      2. Holli K,
      3. Visakorpi T,
      4. Koivula T,
      5. Helin HH,
      6. Isola JJ
      . Association of c-erbB-2 protein over-expression with high rate of cell proliferation, increased risk of visceral metastasis and poor long-term survival in breast cancer. Int J Cancer 1991;49:650–5.
      OpenUrlCrossRefPubMed
      1. Lin NU,
      2. Bellon JR,
      3. Winer EP
      . CNS metastases in breast cancer. J Clin Oncol 2004;22:3608–17.
      OpenUrlAbstract/FREE Full Text
      1. Montemurro F,
      2. Sarotto I,
      3. Casorzo L,
      4. Pisacane A,
      5. Aglietta M,
      6. De RG
      . HER-2 and central nervous system metastasis in patients with breast cancer. Clin Breast Cancer 2004;5:232–4.
      OpenUrlCrossRefPubMed
    32. ↵
      1. Pestalozzi BC,
      2. Brignoli S
      . Trastuzumab in CSF. J Clin Oncol 2000;18:2349–51.
      OpenUrlFREE Full Text
    33. ↵
      1. Zhang GH,
      2. Yang WT,
      3. Zhou XY,
      4. Zeng Y,
      5. Lu HF,
      6. Shi DR
      . Study of the correlation between HER-2 gene and lymphangiogenesis and their prognostic significance in human breast cancer. Zhonghua Yi Xue Za Zhi 2007;87:155–60.
      OpenUrlPubMed
    PreviousNext
    Back to top
    Molecular Cancer Therapeutics: 9 (7)
    July 2010
    Volume 9, Issue 7
    • Table of Contents
    • Table of Contents (PDF)
    • About the Cover

    Sign up for alerts

    View this article with LENS

    Open full page PDF
    Article Alerts
    Sign In to Email Alerts with your Email Address
    Email Article

    Thank you for sharing this Molecular Cancer Therapeutics article.

    NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

    Enter multiple addresses on separate lines or separate them with commas.
    Effective Therapeutic Targeting of the Overexpressed HER-2 Receptor in a Highly Metastatic Orthotopic Model of Esophageal Carcinoma
    (Your Name) has forwarded a page to you from Molecular Cancer Therapeutics
    (Your Name) thought you would be interested in this article in Molecular Cancer Therapeutics.
    CAPTCHA
    This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
    Citation Tools
    Effective Therapeutic Targeting of the Overexpressed HER-2 Receptor in a Highly Metastatic Orthotopic Model of Esophageal Carcinoma
    Stephanie J. Gros, Nina Kurschat, Thorsten Dohrmann, Uta Reichelt, Ana-Maria Dancau, Kersten Peldschus, Gerhard Adam, Robert M. Hoffman, Jakob R. Izbicki and Jussuf T. Kaifi
    Mol Cancer Ther July 1 2010 (9) (7) 2037-2045; DOI: 10.1158/1535-7163.MCT-10-0209

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
    Share
    Effective Therapeutic Targeting of the Overexpressed HER-2 Receptor in a Highly Metastatic Orthotopic Model of Esophageal Carcinoma
    Stephanie J. Gros, Nina Kurschat, Thorsten Dohrmann, Uta Reichelt, Ana-Maria Dancau, Kersten Peldschus, Gerhard Adam, Robert M. Hoffman, Jakob R. Izbicki and Jussuf T. Kaifi
    Mol Cancer Ther July 1 2010 (9) (7) 2037-2045; DOI: 10.1158/1535-7163.MCT-10-0209
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
    • Tweet Widget
    • Facebook Like
    • Google Plus One

    Jump to section

    • Article
      • Abstract
      • Introduction
      • Materials and Methods
      • Results
      • Discussion
      • Disclosure of Potential Conflicts of Interest
      • Acknowledgments
      • Footnotes
      • References
    • Figures & Data
    • Info & Metrics
    • PDF
    Advertisement

    Related Articles

    Cited By...

    More in this TOC Section

    • HPV-16 and Transcriptional Gene Silencing
    • E1A Nanoparticles Enhance Cervical Cancer Radiosensitivity
    • Internalizing Antibodies Targeting Tumor Sphere Cells
    Show more Research Articles
    • Home
    • Alerts
    • Feedback
    • Privacy Policy
    Facebook  Twitter  LinkedIn  YouTube  RSS

    Articles

    • Online First
    • Current Issue
    • Past Issues
    • Meeting Abstracts

    Info for

    • Authors
    • Subscribers
    • Advertisers
    • Librarians

    About MCT

    • About the Journal
    • Editorial Board
    • Permissions
    • Submit a Manuscript
    AACR logo

    Copyright © 2021 by the American Association for Cancer Research.

    Molecular Cancer Therapeutics
    eISSN: 1538-8514
    ISSN: 1535-7163

    Advertisement