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EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging.

Zhou J, Joshi BP, Duan X, Pant A, Qiu Z, Kuick R, Owens SR, Wang TD - Clin Transl Gastroenterol (2015)

Bottom Line: No downstream signaling was observed.We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively.On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT

Objectives: Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears "invisible" on conventional wide-field endoscopy.

Aims: We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging.

Methods: We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence.

Results: After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

Conclusions: We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

No MeSH data available.


Related in: MedlinePlus

Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of (a) QRH*-Cy5.5 peptide (red) co-localizes with that of (b) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in (c) merged image, P=0.71. (d) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for (e) dysplasia and (f) normal. (g) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. (h) Dysplasia (n=29) showed significantly higher fluorescence intensities than normal (n=15) by an average of 19.4-fold, P=1.7 × 10−9 by two-sample t-test on log-transformed data. (i) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.
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fig6: Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of (a) QRH*-Cy5.5 peptide (red) co-localizes with that of (b) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in (c) merged image, P=0.71. (d) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for (e) dysplasia and (f) normal. (g) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. (h) Dysplasia (n=29) showed significantly higher fluorescence intensities than normal (n=15) by an average of 19.4-fold, P=1.7 × 10−9 by two-sample t-test on log-transformed data. (i) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

Mentions: On confocal microscopy, we observed strong binding of QRH*-Cy5.5 (red) and AF488-labeled anti-EGFR (green) to the surface (arrows) of dysplastic colonocytes in human colonic specimens, Figure 6a, b, respectively. Colocalization of peptide and antibody binding can be seen on merged image, P=0.71, Figure 6c. Contrast between dysplasia and normal for peptide binding can be appreciated at the lesion border, Figure 6d. A high-magnification view of white boxes in Figure 6d is shown for dysplasia, Figure 6e, and normal, Figure 6f. Fluorescence intensities were measured from sets of three (dashed white) boxes with dimensions of 30 × 30 μm2 to calculate the target-to-background ratio. Corresponding immunohistochemistry from Figure 6d shows increased reactivity for EGFR in dysplasia, Figure 6g. Dysplasia (n=29) showed a significantly higher mean fluorescence intensity compared with that of normal (n=15) by 19.4-fold, Figure 6h. The receiver operating characteristic curve shows 90% sensitivity and 93% specificity for distinguishing dysplasia from normal with an area under curve of 0.94.


EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging.

Zhou J, Joshi BP, Duan X, Pant A, Qiu Z, Kuick R, Owens SR, Wang TD - Clin Transl Gastroenterol (2015)

Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of (a) QRH*-Cy5.5 peptide (red) co-localizes with that of (b) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in (c) merged image, P=0.71. (d) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for (e) dysplasia and (f) normal. (g) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. (h) Dysplasia (n=29) showed significantly higher fluorescence intensities than normal (n=15) by an average of 19.4-fold, P=1.7 × 10−9 by two-sample t-test on log-transformed data. (i) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4816258&req=5

fig6: Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of (a) QRH*-Cy5.5 peptide (red) co-localizes with that of (b) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in (c) merged image, P=0.71. (d) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for (e) dysplasia and (f) normal. (g) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. (h) Dysplasia (n=29) showed significantly higher fluorescence intensities than normal (n=15) by an average of 19.4-fold, P=1.7 × 10−9 by two-sample t-test on log-transformed data. (i) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.
Mentions: On confocal microscopy, we observed strong binding of QRH*-Cy5.5 (red) and AF488-labeled anti-EGFR (green) to the surface (arrows) of dysplastic colonocytes in human colonic specimens, Figure 6a, b, respectively. Colocalization of peptide and antibody binding can be seen on merged image, P=0.71, Figure 6c. Contrast between dysplasia and normal for peptide binding can be appreciated at the lesion border, Figure 6d. A high-magnification view of white boxes in Figure 6d is shown for dysplasia, Figure 6e, and normal, Figure 6f. Fluorescence intensities were measured from sets of three (dashed white) boxes with dimensions of 30 × 30 μm2 to calculate the target-to-background ratio. Corresponding immunohistochemistry from Figure 6d shows increased reactivity for EGFR in dysplasia, Figure 6g. Dysplasia (n=29) showed a significantly higher mean fluorescence intensity compared with that of normal (n=15) by 19.4-fold, Figure 6h. The receiver operating characteristic curve shows 90% sensitivity and 93% specificity for distinguishing dysplasia from normal with an area under curve of 0.94.

Bottom Line: No downstream signaling was observed.We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively.On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT

Objectives: Colorectal cancer initially lies dormant as dysplasia, a premalignant state that provides an opportunity for early cancer detection. Dysplasia can be flat in morphology, focal in size, and patchy in distribution, and thus it appears "invisible" on conventional wide-field endoscopy.

Aims: We aim to develop and validate a peptide that is specific for epidermal growth factor receptor (EGFR), a cell surface target that is overexpressed in colonic adenomas and is readily accessible for imaging.

Methods: We expressed and purified the extracellular domain of EGFR for use with phage display to identify a peptide QRHKPRE that binds to domain 2 of this target. A near-infrared fluorescence endoscope was used to perform in vivo imaging to validate specific peptide binding to spontaneous colonic adenomas in a mouse model with topical administration. We also validated specific peptide binding to human colonic adenomas on immunohistochemistry and immunofluorescence.

Results: After labeling with Cy5.5, we validated specific peptide binding to EGFR on knockdown and competition studies. Peptide binding to cells occurred within 2.46 min and had an affinity of 50 nm. No downstream signaling was observed. We measured a target-to-background ratio of 4.0±1.7 and 2.7±0.7, for polyps and flat lesions, respectively. On immunofluorescence of human colonic specimens, greater intensity from peptide binding to dysplasia than normal was found with a 19.4-fold difference.

Conclusions: We have selected and validated a peptide that can be used as a specific contrast agent to identify colonic adenomas that overexpress EGFR in vivo on fluorescence endoscopy.

No MeSH data available.


Related in: MedlinePlus