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Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K-ras, BRAF, and PIK3CAgene mutations with tumor resistance to panitumumab alone.

Investigators using anti-EpoR antibodies for immunoblotting and immunostaining have reported erythropoietin receptor (EpoR) expression in nonhematopoietic tissues including human tumors. However, these antibodies detected proteins of 66 to 78 kDa, significantly larger than the predicted molecular weight of EpoR (56-57 kDa). We investigated the specificity of these antibodies and showed that they all detected non-EpoR proteins. C-20 detected 3 proteins in tumor cell lines (35, 66, and 100 kDa). Sequences obtained from preparative gels had similarity to the C-20–immunizing peptide. The 66-kDa protein was a heat shock protein (HSP70) to which antibody binding was abrogated in peptide competition experiments. Antibody M-20 readily identified a 59-kDa EpoR protein. However, neither M-20 nor C-20 was suitable for detection of EpoR using immunohistochemical methods. We concluded that these antibodies have limited utility for detecting EpoR. Thus, reports of EpoR expression in tumor cells using these antibodies should be viewed with caution.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG−/2 mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG−/− mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG−/− mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-κB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG−/− mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

Keratinocyte growth factor (KGF) is emerging asan important mediator of mucosal defense and repair inthe colon. The aim of the present study was to evaluateand further characterize the effects of exogenous KGF administration utilizing the dextran sodiumsulfate (DSS) model of colitis in mice. Colitis wasinduced via oral administration of DSS (5 g/100 ml) toBalb/c mice for eight days. Intraperitonealadministration of KGF (5 mg/kg, once daily) or vehicle (VEH)was initiated 1 hr prior to the induction of the colitis(N = 10, each group). Mucosal injury of the entire colonwas histologically assessed and graded. An approximately fourfold reduction in the cryptdamage score was noted in the KGF group when compared tocontrols (VEH) (2.8 ± 1.03 and 11.4 ±0.78, respectively). The significant reduction ofmucosal injury in KGF treated mice confirms that KGF isa key mediator maintaining the integrity of the colonicmucosa.

The presence of aflatoxin-B1-formamidopyrimidine, a persistent aflatoxin-deoxyribonucleic acid (DNA) adduct, was investigated in vivo by immunohistochemical analysis in 14 paired hepatocellular carcinoma and nontumorous human liver tissue sections using a monoclonal anti-aflatoxin-B1-formamidopyrimidine antibody. Nuclear and cytoplasmic accumulations of adducts were found in 4 of 14 nontumorous specimens but in none of 14 tumorous tissues and in none of three normal control livers. In vitro adduct formation and cellular DNA was investigated with a modified DNA immunoblot assay. These studies revealed (a) no significant difference in the amount of adduct formed by DNA samples with or without integrated hepatitis B virus DNA, (b) no difference in the amount of adduct formed with DNA from either tumorous or nontumorous tissues from a given individual, and (c) remarkable and reproducible differences in the capacity of DNA from different individuals to form in vitro adducts. Our DNA immunoblot assay will facilitate further studies on the molecular role of aflatoxin-B1-formamidopyrimidine adducts in human hepatocarcinogenesis.

EPO stimulates formation of red blood cells by binding to and activating the EPO receptor (EPOR) expressed on the surface of erythroid progenitor cells. Recently EPOR was reportedly expressed in other, non-hematopoietic tissues including brain, kidney, heart and endothelium. EPOR expression has also been reported in tumor tissues and tumor cell lines and many investigators have suggested that EPO may promote tumor growth. This has raised concerns about the use of rHuEPO to treat anemia associated with chemotherapy in cancer patients. To evaluate this, we have examined EPOR expression and binding in tissues and cell lines by quantitative PCR, Western blot analysis and EPO binding assays. Quantitative RT-PCR analysis revealed that the EPOR gene is transcribed in most tissues and cell lines. However non-hematopoietic EPOR transcript levels were lower than that found in hematopoietic cells. No evidence of elevated EPOR gene transcription in tumor vs. non-tumor tissues or in tumor cell lines analyzed was observed. Using Western analysis, we found that commercial anti-EPOR antibodies commonly used to examine EPOR protein expression in published tumor studies, cross-reacted with multiple proteins and/or reacted with proteins of sizes different from that predicted for EPOR. This suggests that in many publications where these antibodies were used, claims of expression of EPOR protein in tumors are of questionable significance. A rabbit polyclonal antibody was developed that specifically reacted with a protein band on Western blots of the size predicted for EPOR. The antibody reacted specifically with a band in COS cells transfected with a human EPOR expressing construct but not in transfection controls. This antibody also reacted with the same sized protein in non-hematopoietic cells. We concluded that EPOR mRNA and EPOR protein were present in most normal cell types examined. Thus the detection of EPOR in tumors and tumor cell lines likely represents the continued expression reflecting the tissue of tumor origin. Since expression does not equate to the presence of surface receptor, binding assays with 125I EPO to non-hematopoietic tumor cell lines were performed. Tumor cell lines showed weak to no detectable EPO binding. These results suggest that although EPOR protein is synthesized in these cells, little or no receptor is expressed on their cell surface. Furthermore, if limited amounts of EPO bound EPOR in tumor lines any biological effect may be different or muted compared to classical responses in hematopoietic cells.