Volume 129, Issue 1, Pages 66-73 (July 2005) Discovery and Identification of α-Defensins as Low Abundant, Tumor-Derived Serum Markers in Colorectal Cancer  Christian Melle, Günther Ernst, Bettina Schimmel, Annett Bleul, Heike Thieme, Roland Kaufmann, Henning Mothes, Utz Settmacher, Uwe Claussen, Karl-Jürgen Halbhuber, Ferdinand von Eggeling  Gastroenterology  Volume 129, Issue 1, Pages 66-73 (July 2005) DOI: 10.1053/j.gastro.2005.05.014 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Box plot of concentration of HNP1–3 in normal and tumor tissue found by ProteinChip arrays (SAX2). Gastroenterology 2005 129, 66-73DOI: (10.1053/j.gastro.2005.05.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Identification of HNP1–3: (A) Normalized ProteinChip arrays profiles of the immunodepletion assays of microdissected colonic tissue. For HNP1–3 identification, colon cancer tissue was used as starting material for immunodepletion assays by the corresponding monoclonal antibody. The negative control was performed without a specific antibody. The peaks at 3.37, 3.44, and 3.49 kilodaltons representing HNP1–3 were detectable in a higher amount in the negative control. (B) Normalized ProteinChip arrays spectra of the immunocapturing assay of microdissected colon carcinoma tissue. HNP1–3 were captured from microdissected colon carcinoma tissue by the corresponding monoclonal antibody bound on IDM beads. The peaks at 3.37, 3.44, and 3.48 kilodaltons representing HNP1–3 were clearly detectable in samples eluted from the IDM beads. In control assays without the specific antibody, no HNP1–3 were captured. (C) Areas with positive and negative reaction in IHC were microdissected and analyzed on ProteinChip arrays. Signals with a molecular mass of 3.37, 3.44, and 3.49 kilodaltons representing HNP1–3 were detectable in protein lysates from positive areas and were absent in the negative areas. Gastroenterology 2005 129, 66-73DOI: (10.1053/j.gastro.2005.05.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Structure of mature human HNP1–3. Amino acid sequence is shown in single letter code, differences in bold letters. Gastroenterology 2005 129, 66-73DOI: (10.1053/j.gastro.2005.05.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Immunohistochemistry (IHC) of HNP1–3 visualized by normal and laser scanning microscopy (LSM; positive reaction is depicted in blue). (A and B) IHC of HNP1–3 on colon carcinoma. (A) Increased signal intensity in carcinoma (Ca) structures (overview with ×25 magnification; N, normal epithelium). (B) Strong positive reaction of invasive growing carcinoma complexes (Ca; magnification of ×120). Gastroenterology 2005 129, 66-73DOI: (10.1053/j.gastro.2005.05.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Box plot of serum concentration of HNP1–3 for controls and patients with colon carcinoma for both independently analyzed sample sets (A and B). Gastroenterology 2005 129, 66-73DOI: (10.1053/j.gastro.2005.05.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 ROC curve for HNP1–3 serum concentration of colon cancer patients and normal controls. The AUC was calculated as 0.77: The sensitivity of 100% is corresponding to a specificity of 69% (A). The independent analysis of the validation set (B) resulted in a P value of .0056. The median for normal controls was 5.60 ng/mL and 14.99 ng/mL for patients. The ROC resulted in a sensitivity of 100% at a specificity of 65.2% and a cut-off of 12.3 ng/mL. The AUC was calculated as 0.840. Gastroenterology 2005 129, 66-73DOI: (10.1053/j.gastro.2005.05.014) Copyright © 2005 American Gastroenterological Association Terms and Conditions