ECP 2023 Abstracts

S307 Virchows Archiv (2023) 483 (Suppl 1):S1–S391 13 E-PS-15-011 Long non-coding RNA NEAT1 cannot be used as a diagnostic and prognostic biomarker in patients with locally advanced rectal cancer K. Eric*, J. Rosic, M. Miladinov, S. Dragicevic, G. Barisic, V. Marko- vic, K. Zeljic *Department of Pathology, Pathohistology and Medical Cytology, Uni- versity Clinical Centre of Serbia, Serbia Background & objectives: The NEAT1 (Nuclear Paraspeckle Assem- bly Transcript 1) gene encodes a long non-coding RNA that is deregu- lated in carcinomas of the gastrointestinal tract. Diagnostic and pre- dictive potential of NEAT1 was investigated in patients with locally advanced rectal cancer. Methods: The study group consisted of 19 patients with rectal cancer treated with neoadjuvant chemoradiotherapy (nCRT). RNA was iso- lated with TRIzol reagent from samples of rectal cancer and noncan- cerous tissue before and after nCRT. The relative expression level of NEAT1 normalised to GAPDH was determined by qRT-PCR method. Results: Expression of NEAT1 did not differ between rectal cancer and noncancerous tissue before nCRT (p=0.953) and cancer and noncan- cerous tissue after nCRT (p=0.210). There was no difference in NEAT1 expression between tumour tissue before and after nCRT (p=0.079). NEAT1 was significantly higher in noncancerous tissue before than after nCRT (p=0.005). Therapy responders (TRG1, TRG2) and non- responders (TRG3, TRG4) did not differ in NEAT1 levels in tumour tissue before (p=0.790) and after nCRT (p=0.352). NEAT1 expression in rectal cancer tissue before nCRT cannot be used as a biomarker to distinguish responders from non-responders (AUC=0.559, 95%CI=0- 1, p=0.790). Demographic and clinicopathological characteristics were not associated with NEAT1 expression in rectal cancer tissue. Conclusion: The obtained results suggest that the long noncoding RNA NEAT1 cannot be considered as a biomarker with diagnostic potential or for predicting response to nCRT in patients with rectal cancer. Validation of the current results in a larger group of patients with locally advanced rectal cancer is warranted. Funding: This work was supported by the Strategic Project of the Ser- bian Academy of Sciences and Arts Molecular basis of response to chemioradiotherapy in rectal cancer - MOHERATEKA, F-69 E-PS-15-012 Stability of testing of microsatellite instability J. Fairley*, S. Deans *GenQA, United Kingdom Background & objectives: Microsatellite instability testing (MSI) is important in determining the potential hereditary nature of colorectal cancer and recently has been expanded to be a biomarker for immune checkpoint inhibitors. This testing needs to be of high quality therefore EQA is important. Methods: GenQA has provided external quality assessment (EQA) for MSI testing for over ten years both as a standalone EQA and as part of the colorectal cancer testing EQA . Participants test formalin-fixed paraffin-embedded tissue and are expected to report their results in their usual format and interpret in the context of the clinical scenario supplied. Results: The standard of MSI testing has been high with an average error rate of less than 2%. The 2022 EQAs error rates were higher at 10% and 15% respectively. For the MSI EQA the errors seemed to be technique related. For the colorectal cancer EQA this was due to an unusual subtle signal patterns which highlighted the limitations of testing without matched germline DNA. Assessment of BRAF variants and MLH1 promoter methylation testing was introduced in 2016 to reflect routine testing strategies for the iden- tification of Lynch syndrome. BRAF variant testing has been accurate, however, errors have been reported by laboratories for MLH1 promoter methylation. Conclusion: The provision of EQA for MSI testing has shown improvements in the quality of testing and interpretation of results by participating laboratories. Issues have been observed when more challenging tests and samples are introduced such as MLH1 promoter methylation testing demonstrating the need for further education and EQA in this evolving field of molecular pathology. E-PS-15-013 Epigenetic analysis of surrogate markers in consensus molecular subtypes of colon cancer D. Guerrero-Setas*, I. Fernández De Los Reyes, I. Monreal- Santesteban, L. Alonso, B. Fernández-Marlasca, P. Azcue, J. Suarez, M. Gómez-Dorronsoro *Hospital Universitario de Navarra, Spain Background & objectives: Consensus Molecular Subtypes (CMS) of colon cancer (CC) are obtained by surrogate immunohistochemical expression of CDX2, FRMD6, HTR2B and ZEB1. Aberrant methyla- tion contributes to tumour progression. The objective was to study the aberrant methylation of the surrogate genes in CC. Methods: 144 stage II (55.6%) and III (44.4%) tumours were classi- fied into CMSs with the primary antibodies against mismatch repair proteins-MMR and proteins of the surrogate panel. CDX2, FRMD6 and ZEB1 methylation levels were analysed by bisulfite pyrosequencing after bisulfite modification of DNA and PCR with specific primers designed for this study. It was not possible to design primers for HTR2B. Results: Tumours were classified into CMS1, CMS2/CMS3 and CMS4 in 18 (12.5%), 117 (81.3%) and 9 patients (6.3%), respec- tively. CDX2 and ZEB1 were hypermethylated in 32.8% and 32.6% of the cases and FRMD6 hypomethylated in 50.9% of the patients. These alterations were more frequent in tumoral than in normal tissue. FRMD6 and ZEB1 IHC expression were not associated with any of the variables tested. Nevertheless, CC cases with absent/low CDX2 expression were associated with CDX2 hypermethylation and were preferentially of mesenchymal type, MMR-defective, stage III, less differentiated and right colon-sided CC tumours (p=0.044, p<0.005, p=0.024, p=0.006, p=0.093, respectively). Conclusion: We describe for the first time the epigenetic abnormali- ties of two surrogate markers used for CMS classification, i.e. ZEB1 hypermethylation and FRMD6 hypomethylation. We also demonstrate that ZEB1 and CDX2 hypermethylation are associated with CMS1 subtype. CDX2 hypermethylation is clearly associated with less CDX2 expression and bad prognostic variables. E-PS-15-014 Usefulness of an extense Next Generation Sequencing panel in endometrial cancer D. Guerrero-Setas*, T. Zudaire, I. Fernández De Los Reyes, Y. Laplaza, P. Fernández, M.C. Caballero, B. Fernández-Marlasca, E. Gochi, M. Gómez, J.C. Muruzabal, R. Guarch *Hospital Universitario de Navarra, Spain Background & objectives: The study of POLE, TP53 and MMR genes by molecular and immunohistochemical techniques are essential for the molecular classification of endometrial cancer (EC). We analyse whether the use of Next-Generation-Sequencing (NGS) gives added value to EC diagnosis. Methods: Targeted DNA/RNA-based NGS was conducted to identify pathogenic/likely pathogenic/uncertain variants in 32 endometrioid and clear-cell EC. DNA/RNA were extracted from macrodissected areas. Library preparation was carried out using a 161-gene panel with