Gastroenterology

Gastroenterology

Volume 138, Issue 2, February 2010, Pages 435-446
Gastroenterology

Imaging and Advanced Technology
In Vivo Molecular Imaging of Colorectal Cancer With Confocal Endomicroscopy by Targeting Epidermal Growth Factor Receptor

https://doi.org/10.1053/j.gastro.2009.10.032Get rights and content

Background & Aims

Epidermal growth factor receptor (EGFR) is a therapeutic target for colorectal cancer (CRC). However, technical challenges have limited in vivo imaging of EGFR in CRCs. Confocal laser endomicroscopy (CLE) enables accurate microscopic visualization of CRC in patients during endoscopy. We evaluated the ability to use CLE in vivo for instantaneous molecular imaging of EGFR in CRC models.

Methods

Tumors were grown in mice (n = 68) from human CRC cell lines that expressed high (SW480 cells) or low (SW620 cells) levels of EGFR. Tumors were visualized in vivo with a handheld CLE probe after injection of fluorescently labeled EGFR antibodies. EGFR-specific fluorescence was graded from 0 to 3+. Neoplastic and non-neoplastic specimens from human colorectal mucosa were examined. In vivo findings were correlated with histopathology, immunohistochemistry, and fluorescence microscopy analyses.

Results

CLE analysis of cell cultures confirmed the different expression levels of EGFR between cell lines. In living animals, CLE differentiated EGFR expression levels between tumor cell limes (mean fluorescence, 1.92 ± 0.22 [SW480] and 0.59 ± 0.21 [SW620], P = .0004). CLE analysis of EGFR expression in human specimens allowed distinction of neoplastic from non-neoplastic tissues (mean fluorescence, 2.0 ± 0.37 vs 0.25 ± 0.16, respectively, P = .0035).

Conclusions

CLE can be used for in vivo, molecular analysis of CRC and to differentiate EGFR expression patterns in xenograft tumors and human tissue samples. Because CLE can be performed during endoscopy, in vivo molecular imaging might be used in diagnosis of CRC and to predict response to targeted therapies.

Section snippets

Tumor Cell Lines

SW480 and SW620 cells32 were cultured in RPMI (with 10% fetal calf serum, 1% glutamine, 1% streptomycin, and 1% penicillin) at 37°C (5% CO2). Cells were harvested after 24–48 hours. For fluorescence-activated cell sorting (FACS), 105 tumor cells were incubated with 2.5 μL of fluorescein isothiocyanate (FITC)-labeled anti-EGFR-antibody (CBL416F; Chemicon/Millipore, Schwalbach, Germany), isotype control (36875K; Pharmingen, Germany), or no antibody (negative control). Immunohistochemistry (IHC)

Differential EGFR Expression by Tumor Cell Lines

FACS analysis confirmed the high EGFR expression of SW480 cells, whereas SW620 cells only showed an absent-to-mild EGFR staining. These patterns were paralleled by a strong specific staining of SW480 cells in IHC of cytospins in contrast to an almost absent specific staining of SW620 cells (Figure 1).

The ability of handheld CLE to discriminate EGFR expression was corroborated by visualizing unfixed tumor cells in cell culture after incubation with FITC-labeled antibody: A strong cellular signal

Discussion

In this study, the first evidence is provided that in vivo endomicroscopic molecular imaging of human CRC is possible by specifically targeting EGFR with a fluorescently labeled antibody. This approach successfully visualized and differentiated human CRC tumor types based on their EGFR expression in a rodent xenograft model in vivo. To mimic a potential topical use of such molecular imaging in CLE in humans, labeled antibody was applied to unfixed human tissue specimens, and successful

Acknowledgments

The authors thank Kathrin Kuhr, Institut für Medizinische Biometrie, Epidemiologie und Informatik, Universitätsmedizin Mainz, Germany, for help with the statistical analysis.

Aspects of this trial are part of the doctor of medicine theses of A.Z. and S.F.

References (54)

  • R. Kiesslich et al.

    Identification of epithelial gaps in human small and large intestine by confocal endomicroscopy

    Gastroenterology

    (2007)
  • E. Endlicher et al.

    Hexaminolevulinate-induced fluorescence endoscopy in patients with rectal adenoma and cancer: a pilot study

    Gastrointest Endosc

    (2004)
  • L. Saltz

    Epidermal growth factor receptor-negative colorectal cancer: is there truly such an entity?

    Clin Colorectal Cancer

    (2005)
  • M. Moroni et al.

    EGFR FISH in colorectal cancer: what is the current reality?

    Lancet Oncol

    (2008)
  • A. Jemal et al.

    Cancer statistics, 2008

    CA Cancer J Clin

    (2008)
  • J.V. Selby et al.

    A case-control study of screening sigmoidoscopy and mortality from colorectal cancer

    N Engl J Med

    (1992)
  • R.S. Herbst et al.

    Monoclonal antibodies to target epidermal growth factor receptor-positive tumors: a new paradigm for cancer therapy

    Cancer

    (2002)
  • F. Ciardiello et al.

    EGFR antagonists in cancer treatment

    N Engl J Med

    (2008)
  • R.I. Nicholson et al.

    EGFR and cancer prognosis

    Eur J Cancer

    (2001)
  • H.S. Abd El All et al.

    Epidermal growth factor receptor in colorectal carcinoma: correlation with clinico-pathological prognostic factors

    Colorectal Dis

    (2008)
  • N.S. Goldstein et al.

    Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system

    Cancer

    (2001)
  • V. Grunwald et al.

    Developing inhibitors of the epidermal growth factor receptor for cancer treatment

    J Natl Cancer Inst

    (2003)
  • G. Galizia et al.

    Epidermal growth factor receptor (EGFR) expression is associated with a worse prognosis in gastric cancer patients undergoing curative surgery

    World J Surg

    (2007)
  • L.B. Saltz et al.

    Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor

    J Clin Oncol

    (2004)
  • D. Cunningham et al.

    Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer

    N Engl J Med

    (2004)
  • D.J. Jonker et al.

    Cetuximab for the treatment of colorectal cancer

    N Engl J Med

    (2007)
  • A. Lievre et al.

    KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab

    J Clin Oncol

    (2008)
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    Conflicts of interest The authors disclose the following: Peter Delaney is director of technology at Optiscan Pty, Ltd, Notting Hill, Victoria, Australia. Ralf Kiesslich and Markus F. Neurath report having received research grants from Pentax, Tokio, Japan. The remaining authors disclose no conflicts.

    Funding Supported by a government grant of the “Stiftung Rheinland-Pfalz für Innovation” (961-38 62 61/821-Projekt 821) (to M.G.).

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