SFEBES2026 Oral Communications Adrenal and Cardiovascular (6 abstracts)
Validation of prognostic biomarkers in adrenocortical carcinoma through Next-Generation Sequencing and pyrosequencing in a real-life setting
Abubaker Mohamed 1 , Lorenzo Tucci 2,3,4 , Dario De Biase 5,6 , Juliane Lippert 7 , Lisa James 1 , Nicola Chadderton 1 , Alice Fair 1 , Amina Mulla 1 , Brendan O’Sullivan 1 , Kassiani Skordilis 8 , Antonio De Leo 4,6 , Phillipe Taniere 1 , Guido Di Dalmazi 3,4 & Cristina L Ronchi 2,9
1Molecular Pathology Diagnostic Service, Queen Elizabeth Hospital Birmingham, University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom; 2Department of Metabolism and Systems Science, University of Birmingham, Birmingham, United Kingdom; 3Division of Endocrinology and Diabetes Prevention and Care, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 4Department of Medical and Surgical Sciences (DIMEC), Alma Mater Studiorum University of Bologna, Bologna, Italy; 5Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy; 6Solid Tumor Molecular Pathology Laboratory, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 7Institute of Human Genetics, University of Wuerzburg, Wuerzburg, Germany; 8Department of Histopathology, Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom; 9Department of Endocrinology, Queen Elizabeth Hospital Birmingham NHS Trust, Birmingham, United Kingdom
Background: Adrenocortical carcinoma (ACC) is a rare malignancy with heterogeneous outcomes. We showed that somatic variants and PAX5 CpG methylation assessed on paraffin-embedded (FFPE) samples can improve prognostic classification.
Aim: To investigate the real-life feasibility of a Next-Generation Sequencing (NGS) and PAX5 pyrosequencing service for ACC prognostic classification in two European centres.
Methods: 53 ACC patients (operated 2002-2025) were divided into a retrospective cohort with known genetic background, an independent validation cohort and a prospective cohort. Tumour DNA was extracted from FFPE tissue and sequenced using a validated NGS panel covering 10 ACC-specific genes. Variant allele frequency (VAF) thresholds were validated between 5%-10%. For PAX5 methylation, 24 samples (2019-2024) were tested using bisulphite-pyrosequencing quantitative assay to assess 7 CpG sites in the promoter region (>25% indicating hypermethylation). Turnaround time (TAT) and cost-effectiveness were also assessed.
Results: After excluding 11 cases with low-quality DNA, 39 samples were analysed. In the retrospective cohort (n = 6), all somatic variants were confirmed, with two additional TERT promoter variants detected. In the validation cohort, 29 variants were found in 19/33 cases, including pathogenic/likely pathogenic variants (53.5% of total findings) in CTNNB1 (9.3%, mean VAF 62.3%), TP53 (23.3%, mean VAF 63.9%), NF1 (16.3%, mean VAF 41.7%) and variants of uncertain significance in TERT, APC, MEN1, NF1, and ZNRF3 (13.9%). In the prospective cohort (n = 3), a TERT promoter variant was identified (c.-124C>T, VAF 66.85%). For PAX5 pyrosequencing, 9/24 samples (2022-2024) showed reliable results with one case being hypermethylated (mean methylation 68.5%). Average TAT was 21 days, with costs of C=350-400/sample for NGS and C=40/sample for pyrosequencing.
Conclusion: The ACC-specific NGS service was reliable, feasible, and cost-effective. PAX5 pyrosequencing was only suitable for samples aged <2-years. Further investigations are required for prospective testing in view of implementation in clinical setting to improve prognostic classification and identify drug targets.
Volume 117
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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