Cyclophilin C-associated protein (CyCAP) knock-out mice spontaneously develop colonic mucosal hyperplasia and exaggerated tumorigenesis after treatment with carcinogen azoxymethane1

<p>Abstract</p> <p>Background</p> <p>The discovery of a "serrated neoplasia pathway" has highlighted the role of hyperplastic lesions of the colon as the significant precursor of colorectal adenocarcinoma. In mice, hyperplasia of the colonic mucosa is a regular phenomenon after a challenge with colonic carcinogens indicating that mucosal hyperproliferation and thickening, even without cytological dysplasia, represents an early pre-malignant change. Cyclophilin C-associated protein (CyCAP) has been described to down-modulate endotoxin signaling in colorectal murine mucosa and is a murine orthologue of the tumor-associated antigen 90 K (TAA90K)/mac-2-binding protein.</p> <p>Methods</p> <p>Female Balb/c wild-type (WT) and CyCAP knock-out (KO) mice (6–8 weeks old) were administered 2 or 6 weekly subcutaneous injections of azoxymethane. The animals were evaluated post-injection at six weeks for aberrant crypt foci (ACF) study and at five months for colon tumor measurement. The thickness of the colon crypts was measured in microns and the number of colonocytes per crypt was also determined in well-oriented crypts. Morphometric analyses of the colon mucosa were also performed in untreated 6–8 weeks old KO and WT animals. Formalin-fixed/paraffin-embedded colon sections were also studied by immunohistochemistry to determine the Ki-67 proliferation fraction of the colon mucosa, β-catenin cellular localization, cyclin D1, c-myc, and lysozyme in Paneth cells.</p> <p>Results</p> <p>Cyclophilin C-associated protein (CyCAP)^-/-mice, spontaneously developed colonic mucosal hyperplasia early in life compared to wild-type mice (WT) (p < 0.0001, T-test) and crypts of colonic mucosa of the (CyCAP)^-/-mice show higher proliferation rate (p = 0.039, Mann-Whitney Test) and larger number of cyclin D1-positive cells (p < 0.0001, Mann-Whitney Test). Proliferation fraction and cyclin D1 expression showed positive linear association (p = 0.019, Linear-by-Linear Association). The hyperplasia was even more pronounced in CyCAP^-/-mice than in WT after challenge with azoxymethane (p = 0.005, T-test). The length of the crypts (r = 0.723, p = 0.018, Spearman Correlation) and the number of colonocytes per crypt (r = 0.863, p = 0.001, Spearman Correlation) in non-tumorous areas were positively associated with azoxymethane-induced number of tumors. CyCAP^-/-developed larger numbers of tumors than WT animals (p = 0.003, T-Test) as well as overall larger tumor mass (p = 0.016, T-Test). Membranous β-catenin was focally overexpressed in KO mice including proliferative zone of the crypts.</p> <p>Conclusion</p> <p>CyCAP^-/-represent the first described model of spontaneous colonic mucosal hyperplasia. We conclude that CyCAP-deficient mice spontaneously and after challenge with carcinogen develop significantly more colorectal mucosal hyperplasia, an early stage in murine colonic carcinogenesis.</p

Serrated Lesions of the Colorectum: Review and Recommendations From an Expert Panel

Serrated lesions of the colorectum are the precursors of perhaps one-third of colorectal cancers. Cancers arising in serrated lesions are usually in the proximal colon, and account for a disproportionate fraction of cancer identified after colonoscopy

Fli-1 expression in malignant melanoma

Friend leukemia integration site 1 (Fli-1) has been reported as the first nuclear marker of endothelial differentiation; it is expressed in leukocytes and recently demonstrated in melanomas. Formalin-fixed, paraffinembedded tissue sections from 97 melanomas including 69 cases of primary and 28 metastatic melanomas were evaluated by immunohistochemistry. Five melanoma cell lines were evaluated by Western blot and immunocytochemistry. Fli-1 expression was observed in all cell lines. Fli-1 expression was higher in metastatic than in primary tumors (r=0.208, p=0.041, Spearman correlation), it positively correlated with Ki-67 expression (r=0.233, p=0.022, Spearman correlation), and the presence of an ulcer in the primary tumor (r=0.267, p=0.030, Spearman correlation). Therefore, the expression of Fli-1 in malignant melanoma appears to be associated with biologically more aggressive tumors

Simultaneous Imaging and Therapy Using Epitope-Specific Anti-Epidermal Growth Factor Receptor (EGFR) Antibody Conjugates

Matuzumab and nimotuzumab are anti-EGFR monoclonal antibodies that bind to different epitopes of domain III of EGFR. We developed 89Zr-matuzumab as a PET probe for diagnosis/monitoring of response to treatment of a noncompeting anti-EGFR nimotuzumab antibody drug conjugate (ADC) using mouse colorectal cancer (CRC) xenografts. We developed 89Zr-matuzumab and performed quality control in EGFR-positive DLD-1 cells. The KD of matuzumab, DFO-matuzumab and 89Zr-matuzumab in DLD-1 cells was 5.9, 6.2 and 3 nM, respectively. A competitive radioligand binding assay showed that 89Zr-matuzumab and nimotuzumab bound to noncompeting epitopes of EGFR. MicroPET/CT imaging and biodistribution of 89Zr-matuzumab in mice bearing EGFR-positive xenografts (HT29, DLD-1 and MDA-MB-231) showed high uptake that was blocked with pre-dosing with matuzumab but not with the noncompeting binder nimotuzumab. We evaluated nimotuzumab-PEG6-DM1 ADC in CRC cells. IC50 of nimotuzumab-PEG6-DM1 in SNU-C2B, DLD-1 and SW620 cells was dependent on EGFR density and was up to five-fold lower than that of naked nimotuzumab. Mice bearing the SNU-C2B xenograft were treated using three 15 mg/kg doses of nimotuzumab-PEG6-DM1, and 89Zr-matuzumab microPET/CT was used to monitor the response to treatment. Treatment resulted in complete remission of the SNU-C2B tumor in 2/3 mice. Matuzumab and nimotuzumab are noncompeting and can be used simultaneously

Standardization of negative controls in diagnostic immunohistochemistry: recommendations from the international Ad Hoc expert panel

Standardization of controls, both positive and negative controls, is needed for diagnostic immunohistochemistry (dIHC). The use of IHC-negative controls, irrespective of type, although well established, is not standardized. As such, the relevance and applicability of negative controls continues to challenge both pathologists and laboratory budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear which types of positive and negative controls are applicable and/or useful in day-to-day clinical practice. There is a perceived need to provide "best practice recommendations" for the use of negative controls. This perception is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially when IHC is performed for predictive markers, the number of which is rising as personalized medicine continues to develop. Herein, an international ad hoc expert panel reviews classification of negative controls relevant to clinical practice, proposes standard terminology for negative controls, considers the total evidence of IHC specificity that is available to pathologists, and develops a set of recommendations for the use of negative controls in dIHC based on "fit-for-use" principles