Optimisation of osteosarcoma sample processing for epigenetic analysis: A comparative evaluation of formalin-fixed, paraffin-embedded samples with and without decalcification versus fresh tissue

INTRODUCTION: Osteosarcomas (OS), the most common primary malignant bone tumours, are classified as low-grade (characterised by MDM2 amplification) or high-grade (with complex karyotypes). Accurate diagnosis is essential for treatment and prognosis. This study evaluates pre-analytical variables associated with the success or failure of epigenetic analyses in osteosarcoma samples and proposes a standardised preparation protocol. METHODS: Retrospective cohort study of adult patients with OS diagnosed at our sarcoma reference centre (CSUR) over the past 20 years. Pre-analytical variables: year of diagnosis, histological subtype, tissue type, site of origin, sample type (core needle biopsy or surgical specimen with or without chemotherapy), decalcification method (none, EDTA, or nitric acid), and FISH availability. Five 5-μm sections were obtained from each paraffin block. DNA methylation profiling was performed using the Infinium MethylationEPIC v2.0 platform (Illumina). Univariate and multivariate analyses were performed to identify failure predictors. RESULTS: A total of 103 samples from 79 patients were analysed: 58 conventional OS, 14 extraskeletal, 24 parosteal, and 7 dedifferentiated OS. Of the 95 formalin-fixed, paraffin-embedded (FFPE) samples, 43 (45.2%) were suitable for epigenetic analysis, whereas all frozen samples were adequate (100%). Decalcification affected success rates, although not significantly: nitric acid was associated with the highest failure rate (68.97%), followed by EDTA (57.14%) and non-decalcified samples (46.15%). CONCLUSION: FFPE samples are suitable for epigenetic studies, although performance depends on pre-analytical factors. Frozen tissue remains the gold standard. Nitric acid should be avoided. A protocol is proposed that prioritises frozen tissue, documents decalcification methods, excludes strong acids, incorporates quality control measures, and favours samples less than five years old.