1. TUMOUR HISTOLOGY SERUM BIOCHEMISTRY miRNA PROFILE TUMOUR RADIOLOGY FURTHER WORK & CLINICAL APPLICATION SMALL RNA LIBRARY CONSTRUCTION MOLECULAR BIOLOGY & METHODS AIM SURGICAL TREATMENT INTRODUCTION & CLINICAL PRESENTATION Diagnosis, treatment and genome-wide microRNA profiling of a phosphaturic mesenchymal tumour Phosphaturic mesenchymal tumour is an extremely rare tumour of soft tissue and bone. Although a benign neoplasm it can produce severe paraneoplastic effects such as hypophosphatemia and tumour-induced osteomalacia – resulting in chronic bone pain, fractures, muscle pain and fatigue. It causes this through inappropriate secretion of FGF23. A 72 year old male patient was referred to our specialist clinic after a six-year period of general deterioration, “explosive” whole-body pain, decline to disability and loss of vision. Darrell Green 1 | Matthew Stocks 2 | Irina Mohorianu 3 | Martina Billmeier 3 | Ping Xu 3 | Jeremey Turner 4 Andoni Toms 5 | John Nolan 6 | Laszlo Igali 7 | Richard Ball 7 | Tamas Dalmay 3 | William Fraser 1,4 1 Norwich Medical School | 2 School of Computing Sciences | 3 School of Biological Sciences | University of East Anglia 4 Endocrinology | 5 Norwich Radiology Academy | 6 Orthopaedics & Trauma | 7 Histopathology | Norfolk and Norwich University Hospital Correspondence: d.green@uea.ac.uk Figure 1. Positron Emission Tomography (PET) showing tumour location. Radiotracer uptake is seen in the left femoral head. Indium-111 octreotide is a commonly used radiolabel in nuclear medicine for imaging neuroendocrine tumours. The authors would like to thank the patient for the generous donation of tissue samples to the Norwich Biorepository. Adjacent Tumour Figure 3. Small RNA libraries using HD adapters (8% PAGE). The red arrow points to the PCR products of the cDNA library. The black arrow points to HD adapter dimer PCR products. Figure 4. miRNA profile of phosphaturic mesenchymal tumour. miRNA stem loops show mature miRNA (blue) and miRNA* (red). There are 16 downregulated tumour-suppressor miRNAs in this case. BIOLOGICAL ROLE OF FGF23 Increase renal phosphate wasting. Increase phosphate release from bone. Decrease phosphate absorption in the gut. Suppression of 1α-hydroxylase – the renal enzyme responsible for conversion of 25(OH) Vitamin D to 1,25(OH) 2 Vitamin D. Phosphate = 0.4 mmol/L (Normal: 0.8 – 1.5 mmol/L) Alkaline Phosphatase (ALP) = 207 U/L (Normal: 10 – 50 U/L) 1,25(OH) 2 Vitamin D = 31 pmol/L (Normal: 43 – 143 pmol/L) FGF23 = 438 RU/mL (Normal: <100 RU/mL) The tumour was resected by total hip replacement. Patient mobility restored and pain-free in a matter of days. Phosphate = 0.75 mmol/L ALP = 192 U/L 1,25(OH) 2 Vitamin D = 273 pmol/L FGF23 = 44 RU/L Total RNA extracted from tumour and adjacent tissue. Small RNA library construction with HD adapters. Next generation sequencing at The Genome Analysis Centre. Bioinformatic analysis with The UEA Small RNA Workbench. Figure 2. Histology sections of tumour biopsy. H&E staining showing a thickened trabecular bone with an increase of woven and lamellar bone. There is a heterogenous mix of osteoclast-like giant cells and spindled cells. Nuclei are hyperchromatic. Necrosis and haematopoietic activity not present. These findings are consistent with a phosphaturic mesenchymal tumour. microRNAs (miRNAs) are a class of small non-coding RNA molecules that are powerful regulators of gene expression in a mechanism called “RNA silencing”. With >1,000 human miRNAs each specific to a disease, they can be profiled as biomarkers and targets of gene therapy. We aimed to create the first “miRNA profile” of this tumour. Validate miRNA profile using digital PCR and northern blot. Carry out miRNA profiling on other tumours for biological validity. Differentially expressed miRNAs may be used as biomarkers of tumour presence. Downregulated miRNAs may be rescued by LNA therapy, restoring the neoplastic cells to a normal phenotype.