(P 02) METHYLATION PROFILING AS A DIAGNOSTIC ADJUNCT FOR UNDIFFERENTIATED SARCOMAS: A REAL-WORLD EVALUATION

Objective: Aberrant DNA methylation is a key epigenetic mechanism and hallmark of malignancy that offers diagnostic potential through tumor-specific methylation signatures. A recently developed, publicly available sarcoma methylation classifier offers potential diagnostic improvement. We assessed its performance with two aims: (1) to evaluate concordance between methylation-based classification and expert pathology; and (2) to determine whether the classifier could provide refined subtyping.

Methods: DNA was extracted and processed per manufacturer protocol from formalin-fixed, paraffin-embedded (FFPE) sarcoma specimens and run in the Infinium MethylationEPIC v2.0 BeadChip. Methylation data were analyzed via Illumina GenomeStudio and classified using the Heidelberg Epignostix Sarcoma Classifier (v12.3). Classification scores were stratified as high (≥0.90), intermediate (0.30–0.89), or indeterminate ( < 0.30). Methylation results were compared to prior expert pathology and discrepancies categorized as major (clinical impact) or minor (no expected consequence).

Results: Fifty-five sarcoma specimens (2012–2022) were included, predominantly undifferentiated/pleomorphic sarcomas (n=33; 60%) and high-grade (grade 2/3, n=45; 82%). Of 55 samples, 50 (91%) yielded usable methylation profiles (score ≥0.30). Among these, 42 (84%) had high-confidence classifications (≥0.90), and 8 (16%) were intermediate. In the intermediate group, 75% showed concordance with expert pathology; 2 cases had minor discrepancies. In the high-confidence group, 76% were concordant with 5 cases each of major and minor discrepancies.

Major discrepancies included reclassification of tumors initially diagnosed as undifferentiated sarcoma to alveolar rhabdomyosarcoma, schwannoma, or angiomatoid fibrous histiocytoma. In some cases, methylation profiling provided more specific subtypes (e.g., malignant peripheral nerve sheath tumor vs. undifferentiated sarcoma), while in others, expert pathology was more definitive (e.g., malignant PEComa labeled as undifferentiated by classifier).

Conclusion: Methylation profiling showed ~75% concordance with expert pathology and occasionally enabled diagnostic refinement. Compared to DNA/RNA sequencing, it is faster and more cost-effective. Limitations include inflammatory contamination and classifier immaturity in rare subtypes. A prospective study is planned to assess its integration into diagnostic workflows for ambiguous sarcomas.

Demographic summaries from included patients with sarcoma