br Materials and methods br

Materials and methods
Results
Discussion Utilizing IHC, the majority of OS samples were positive for RANKL (68%), with RANKL staining detectable in OS tumor exactly and tumor anaplastic cells. However, a wide range of RANKL expression within the OS component was observed and many samples had low intensity RANKL signal or demonstrated no detectable RANKL staining. Clearly RANKL expression was not uniformly observed within an OS tumor, as only a few samples (16/56) demonstrated more than 10% of the tumor cells positive for RANKL signal. The intensity and frequency of RANKL staining in OS samples observed in this study were substantially less than that observed in GCTB samples using the same antibody for IHC evaluation [23]. The overall incidence of RANKL staining observed in the present study of human OS (68%) is similar to that observed by Lee et al. [34] using a goat polyclonal RANKL antisera (75%) but differs substantially to the 9% RANKL positivity observed by Bago-Horvath [22]. The relatively small sample size (n=56) and limited patient follow-up data precluded any prognostic association with RANKL incidence or expression levels in the present study. We conclude that RANKL is observed in the majority of OS cases although the fraction of positive tumor cells and staining intensity is generally very low. The absence of RANK expression in OS tumor cells observed in the present study is in marked contrast to the two reports that RANK is highly expressed in the majority of primary human OS [20,22]. Mori et al. (2007) reported RANK expression to be homogeneously expressed (in 100% of tumor cells) in 57% of OS biopsy specimenss and Bago-Horvath [22] reported moderate or strongly positive RANK expression in 69% of OS tumor samples. Given that the IHC scoring systems used in all studies were not normalized, one cannot make an unbiased comparison of RANK expression incidence and intensity levels observed in those two studies with the current study. However, it is important to point out distinct features of the present study with the two previously published studies of RANK expression in human OS to address the very high incidence observed in those studies. Firstly, both previously published reports relied on commercially available anti-RANK antibodies and did not provide any evidence for the specificity and reproducibility of staining for IHC applications nor confirmation of RANK expression using independent methods or reagents. Secondly, the use of mAbs for expression analysis in the present study avoids the technical pitfalls of run-to-run reproducibility inherent in polyclonal antibodies. Thirdly, neither previously-published study documented the expected RANK staining within normal myeloid cells in the tumor mass or at the bone/tumor interface within any OS sample to document RANK expression in the expected cell types and provide context for the signal to noise for the reagents and methods used. The validation of the RANK antibodies used for IHC demonstrated concordant positive (and negative) signals between multiple independent methodologies and addressed sensitivity down to fewer than 1800 receptors/cell. Furthermore, in our analysis of OS samples, two independent anti-RANK mAbs, each recognizing distinct epitope binding sites demonstrated an identical positive and negative staining pattern, essentially cross-validating one another. Different alternatively-spliced variants of the human RANK gene have been described which alter exons 7, 8, or 9 encoding in either the transmembrane or cytoplasmic regions of RANK [32], potentially impacting IHC detection. However, the antibodies N-1H8 and N-2B10 bind to the extracellular portion of RANK common to each variant, thus the IHC results reported here would be inclusive of any rare, alternatively-spliced forms. Altogether, these specificity and sensitivity controls, along with the observed expression of RANK in the expected osteoclasts and related cells, substantiates the observation that OS tumor cells do not express RANK to any major degree. It would seem likely that technical limitations to these previously published studies may have accounted for the reportedly high incidence and high expression levels of RANK within OS tumors.