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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

Validation of colonic dysplasia on pathology. (a) Excised colon from Figure 4 shows locations of flat lesions and polyp (dashed red lines), Bar = 2 mm. (b) Histology (H&E) of flat lesion shows nonpolypoid mucosal morphology and foci of low-grade adenomatous dysplasia (red boxes) separated by intervening regions of normal mucosa, Bar=500 μm. (c) Greater mean fluorescence intensities from polyps (n=15) and flat lesions (n=15) were found compared with those from adjacent normal mucosa, T/B ratio 4.0±1.7 and 2.7±0.7, respectively. For polyps, mean±s.d. of the T/B ratio (log2) for QRH*-Cy5.5 and PEH*-Cy5.5 was 1.90±0.60 and 0.62±0.77, P=4.1 × 10−4 by paired, two-sided t-test, respectively, and mean fold difference was 2.43. For flat lesions, the results were 1.39±0.34 and 0.36±0.47, P=7.4 × 10−6 by paired, two-sided t-test, and mean fold difference was 2.05. (d–f) Magnified view of red boxes in b shows histological features of low-grade dysplasia (arrows). (g) Histology (H&E) of polyp along vertical red line in a shows identical histological features of dysplasia. H&E, hematoxylin and eosin; T/B, target-to-background.
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fig5: Validation of colonic dysplasia on pathology. (a) Excised colon from Figure 4 shows locations of flat lesions and polyp (dashed red lines), Bar = 2 mm. (b) Histology (H&E) of flat lesion shows nonpolypoid mucosal morphology and foci of low-grade adenomatous dysplasia (red boxes) separated by intervening regions of normal mucosa, Bar=500 μm. (c) Greater mean fluorescence intensities from polyps (n=15) and flat lesions (n=15) were found compared with those from adjacent normal mucosa, T/B ratio 4.0±1.7 and 2.7±0.7, respectively. For polyps, mean±s.d. of the T/B ratio (log2) for QRH*-Cy5.5 and PEH*-Cy5.5 was 1.90±0.60 and 0.62±0.77, P=4.1 × 10−4 by paired, two-sided t-test, respectively, and mean fold difference was 2.43. For flat lesions, the results were 1.39±0.34 and 0.36±0.47, P=7.4 × 10−6 by paired, two-sided t-test, and mean fold difference was 2.05. (d–f) Magnified view of red boxes in b shows histological features of low-grade dysplasia (arrows). (g) Histology (H&E) of polyp along vertical red line in a shows identical histological features of dysplasia. H&E, hematoxylin and eosin; T/B, target-to-background.

Mentions: After completion of imaging, the animals were killed. The colon was excised and divided longitudinally. The gross specimen with the polyp and flat lesions from Figure 4 are shown, Figure 5a. Fluorescence imaging was used to guide a perpendicular section of the mucosal surface along the horizontal and vertical red lines. The flat lesions show mucosa with nonpolypoid morphology on histology (H&E), Figure 5b. Foci of dysplasia (red boxes) can be seen separated by regions of normal. The average fluorescence intensity from three regions of interest with dimensions of 25 × 25 pixels were picked at random from areas of “high” intensity and adjacent areas of “low” intensity. The target-to-background ratios were determined by taking ratios of the means of these results. We measured significantly greater fluorescence intensity from both polyps (n=15) and flat lesions (n=15) compared with that from the adjacent normal mucosa, target-to-background ratio 4.0±1.7 and 2.7±0.7, respectively. The difference in results between QRH*-Cy5.5 and PEH*-Cy5.5 was significant. A high-magnification view of histology from the flat regions (red boxes) show features of low-grade dysplasia (arrows), including collections of irregular crypts lined by epithelium with crowded, elongated, and hyperchromatic nuclei, Figure 5d–f. Histology of the polyp also shows identical features of dysplasia, Figure 5g.


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)

Validation of colonic dysplasia on pathology. (a) Excised colon from Figure 4 shows locations of flat lesions and polyp (dashed red lines), Bar = 2 mm. (b) Histology (H&E) of flat lesion shows nonpolypoid mucosal morphology and foci of low-grade adenomatous dysplasia (red boxes) separated by intervening regions of normal mucosa, Bar=500 μm. (c) Greater mean fluorescence intensities from polyps (n=15) and flat lesions (n=15) were found compared with those from adjacent normal mucosa, T/B ratio 4.0±1.7 and 2.7±0.7, respectively. For polyps, mean±s.d. of the T/B ratio (log2) for QRH*-Cy5.5 and PEH*-Cy5.5 was 1.90±0.60 and 0.62±0.77, P=4.1 × 10−4 by paired, two-sided t-test, respectively, and mean fold difference was 2.43. For flat lesions, the results were 1.39±0.34 and 0.36±0.47, P=7.4 × 10−6 by paired, two-sided t-test, and mean fold difference was 2.05. (d–f) Magnified view of red boxes in b shows histological features of low-grade dysplasia (arrows). (g) Histology (H&E) of polyp along vertical red line in a shows identical histological features of dysplasia. H&E, hematoxylin and eosin; T/B, target-to-background.
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Related In: Results  -  Collection

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fig5: Validation of colonic dysplasia on pathology. (a) Excised colon from Figure 4 shows locations of flat lesions and polyp (dashed red lines), Bar = 2 mm. (b) Histology (H&E) of flat lesion shows nonpolypoid mucosal morphology and foci of low-grade adenomatous dysplasia (red boxes) separated by intervening regions of normal mucosa, Bar=500 μm. (c) Greater mean fluorescence intensities from polyps (n=15) and flat lesions (n=15) were found compared with those from adjacent normal mucosa, T/B ratio 4.0±1.7 and 2.7±0.7, respectively. For polyps, mean±s.d. of the T/B ratio (log2) for QRH*-Cy5.5 and PEH*-Cy5.5 was 1.90±0.60 and 0.62±0.77, P=4.1 × 10−4 by paired, two-sided t-test, respectively, and mean fold difference was 2.43. For flat lesions, the results were 1.39±0.34 and 0.36±0.47, P=7.4 × 10−6 by paired, two-sided t-test, and mean fold difference was 2.05. (d–f) Magnified view of red boxes in b shows histological features of low-grade dysplasia (arrows). (g) Histology (H&E) of polyp along vertical red line in a shows identical histological features of dysplasia. H&E, hematoxylin and eosin; T/B, target-to-background.
Mentions: After completion of imaging, the animals were killed. The colon was excised and divided longitudinally. The gross specimen with the polyp and flat lesions from Figure 4 are shown, Figure 5a. Fluorescence imaging was used to guide a perpendicular section of the mucosal surface along the horizontal and vertical red lines. The flat lesions show mucosa with nonpolypoid morphology on histology (H&E), Figure 5b. Foci of dysplasia (red boxes) can be seen separated by regions of normal. The average fluorescence intensity from three regions of interest with dimensions of 25 × 25 pixels were picked at random from areas of “high” intensity and adjacent areas of “low” intensity. The target-to-background ratios were determined by taking ratios of the means of these results. We measured significantly greater fluorescence intensity from both polyps (n=15) and flat lesions (n=15) compared with that from the adjacent normal mucosa, target-to-background ratio 4.0±1.7 and 2.7±0.7, respectively. The difference in results between QRH*-Cy5.5 and PEH*-Cy5.5 was significant. A high-magnification view of histology from the flat regions (red boxes) show features of low-grade dysplasia (arrows), including collections of irregular crypts lined by epithelium with crowded, elongated, and hyperchromatic nuclei, Figure 5d–f. Histology of the polyp also shows identical features of dysplasia, Figure 5g.

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