ABSTRACT
OBJECTIVE: To analyse the function of nucleotide pyrophosphatase phosphodiesterase (NPP1), a member of the pyrophosphate pathway, in osteoarthritis (OA). METHODS: mRNA expression of NPP1, ANK ankylosing protein and tissue non-specific alkaline phosphatase was assessed by quantitative PCR. NPP1 protein levels were analysed in mouse and human cartilage samples. Bone metabolism was analysed by F18-positron emission tomography-scanning and µCT in ttw/ttw mice. Ttw/ttw mice are mice carrying a loss-of-function mutation in NPP1. Calcification of articular cartilage was assessed using von Kossa staining and OA severity using the Mankin score. Cartilage remodelling was investigated by type X collagen immunohistochemistry. RESULTS: Expression of NPP1, but not the other members of this pathway, inversely correlated with cartilage calcification and OA severity in mouse and humans. Proinflammatory cytokines downregulated the expression of NPP1, demonstrating an influence of inflammation on matrix calcification. Ttw/ttw mutant mice, carrying a loss-of-function mutation in NPP1, exhibit increased bone formation process in joints compared with wild types. Ttw/ttw mice also developed spontaneous OA-like changes, evaluated by histological analysis and in vivo imaging. Ectopic calcifications were associated with increased expression of collagen X in the cartilage. CONCLUSION: The authors conclude that OA is characterised by the reactivation of molecular signalling cascades involving proinflammatory cytokines, thereby regulating the pyrophosphate pathway which consequently leads to cartilage ossification, at least in part resembling endochondral ossification.
Subject(s)
Arthritis, Experimental/metabolism , Calcinosis/metabolism , Cartilage, Articular/metabolism , Osteoarthritis, Knee/metabolism , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Arthritis, Experimental/pathology , Biomarkers/metabolism , Calcinosis/pathology , Cartilage, Articular/pathology , Collagen Type X/metabolism , Gene Expression Regulation, Enzymologic , Humans , Mice , Mice, Knockout , Osteoarthritis, Knee/pathology , Osteoarthritis, Knee/surgery , Phosphate Transport Proteins/genetics , Phosphate Transport Proteins/metabolism , Phosphoric Diester Hydrolases/genetics , Pyrophosphatases/genetics , RNA, Messenger/metabolism , Severity of Illness Index , Signal Transduction , Stifle/metabolism , Stifle/pathologyABSTRACT
BACKGROUND: HER-2 is the target for antibody-based treatment of breast cancer (trastuzumab), which is highly successful in both advanced disease and the adjuvant setting. HER-2 can be analysed by fluorescence in situ hybridisation (FISH) for gene amplification or immunohistochemistry (IHC) for protein overexpression. AIM: As both methods are known to be influenced by previous tissue processing, to analyse the applicability of both FISH and IHC to decalcified bone metastases of breast cancer. METHODS: A tissue microarray (TMA) was constructed from 149 breast cancer bone metastases. Consecutive TMA sections were analysed by FISH (PathVysion) and IHC (HercepTest). RESULTS: FISH analysis was interpretable in 113 (85.0%) cases. Amplification was seen in 14 (12.4%) interpretable metastases. HER-2 positivity on IHC analysis was 3+ in 9.8% of cases and 2+ in 11.3%. A comparison of the two techniques revealed high concordance. Of the 14 cases of amplification, 10 (71%) showed 3+ IHC staining, two (14%) showed 2+, one (7%) showed 1+, and one (7%) showed 0+. Three of the four amplified cases that did not show 3+ IHC staining had an equivocal FISH result, with a HER-2/centromere 17 ratio of 1.8-2.2. Of the 13 cases that showed IHC 3+ staining, amplification was present in 10 (77%). CONCLUSIONS: HER-2 FISH analysis has an excellent success rate in highly standardised EDTA-decalcified bone metastases, suggesting that this method is easily applicable to decalcified tissues. The high concordance between IHC and FISH suggests that HER-2 IHC may be equally applicable to EDTA-treated tissues as to the usual formalin-fixed tissues.
