Fibrous Dysplasia (FD) is a genetic skeletal disorder characterized by thinning of cortical bone and replacement of the bone marrow with fibro-osseous tissue. The underlying defect in fibrous dysplasia is a mutation of the GNAS gene, which leads to constitutive activation of gene products that interfere with the maturation of osteoprogenitor cells and lead to deposition of abnormal bone. Two studies conducted so far have been performed on the mRNA expression analysis of human fibrous dysplastic bone tissues employing microarray approach to explore the key molecule events in FD development. The present proposal aims at generating mRNA expression profiling of FD clinical samples stored in formalin-fixed paraffin embedded (FFPE) blocks using NanoString platform technology. This platform hybridizes fluorescent barcodes directly to specific nucleic acid sequences, allowing for the nonamplified measurement of up to 800 targets within one sample, working well with FFPE samples. The specific aims of this proposal include: 1) the identification of genes that are differently expressed in fibrous versus non-fibrous human bone in FFPE paraffin blocks; 2) the description of relationships between these genes using multivariate data analysis to search for gene clusters which separate and can characterize the healthy state from the pathologic one; 3) the assesment that NanoString technology has the potential to penetrate into clinical diagnostic practice proving to be robust and reliable for quantification of RNA species extracted from Fibrous Dysplasia clinical samples stored in FFPE blocks. This approach will eventually lead to develop relevant molecular biomarkers to monitor the efficacy of treatments and/or disease progression. Finally the translation of state-of-the-art technology like NanoString approach into quantifiable patient benefits such as the development of effective biomarkers could be extremely attractive also into clinical settings other than FD.
Histopathologic diagnoses and treatment of FD can be improved with molecular testing, such as mRNA expression profiling. Preferably, molecular diagnostics should fit into standard workflows for biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing.
A key constraint of translational research within a rare disease, like FD, study/diagnosis setting is that there is often a limited amount of tissue from which to carry out molecular profiling analyses. Further, starting samples are frequently archival and stored in formalin-fixed paraffin embedded (FFPE) blocks. So in many cases traditional methods of gene expression analysis have strong limitations for clinical application. For example, RT-PCR measures the expression of one gene at a time, and multiplex expression profiling techniques such as microarrays, covering many thousands of transcripts, are often expensive and lack flexibility and reproducibility when evaluating low-quality RNA samples such as those from FFPE.
The Nanostring Technologies nCounter platform is a relatively new technology and has been used within various clinical and research applications (Kim ST et al 2015).
This technology has the potential to penetrate into clinical laboratory diagnostic practice because it has proven to be robust and reliable for quantification of RNA species even extracted from FFPE and, therefore, might be a suitable platform for a gene expression based clinical profiling.
We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE Fibrous Dysplasia samples.
Kim ST, Do IG, Lee J, Sohn I, Kim KM, Kang WK.¿The NanoString-based multigene assay as a novel platform to screen EGFR, HER2, and MET in patients with advanced gastric cancer.¿ Clin Transl Oncol. 2015 Jun;17(6):462-8.