ECP 2023 Abstracts

S317 Virchows Archiv (2023) 483 (Suppl 1):S1–S391 13 and distant metastatis in univariate analysis. Although, a high fre- quency of mutation was observed in high grade tumours, statistical significance was not found. The identification of this significant alteration may demonstrate that it can be used for differential diagnosis in routine practice and also might be biomarker for favourable overall survival of MECs. E-PS-15-046 Development and validation of assays for testing EGFR amplifica- tion in tissues, based on gene copies, RNA and protein expression S. Tsuriel*, V. Hannes, D. Hershkovitz *Tel Aviv Sourasky medical centre, Israel Background & objectives: EGFR amplification is leading to uncon- trolled cell growth and tumour proliferation; it can be used as a bio- marker for treatment based on EGFR inhibition. Still it’s unclear what the best marker for EGFR amplification and how to measure it accurately. Methods: The aim of this study was to develop and validate a proto- col for measuring EGFR gene copy, RNA, and protein expression. A multiplexed digital PCR-based method was developed and validated to detect EGFR gene copy number amplification. Immunohistochemistry was used to assess the protein levels, and qPCR was used to measure EGFR gene expression. Results: A total of 123 clinical FFPE tissue samples from various tumours were collected. The digital PCR-based assay was validated against samples with a known amplification status and found to have 100% agreement. Five different protocols were compared to optimize the immunohistochemistry EGFR labelling. Samples that showed gene copy amplification had high RNA and protein expression, while sam- ples with high RNA also showed high protein levels. However, samples with high EGFR protein levels did not necessarily have a high gene copy number. Conclusion: Efficient and useful methods for measuring EGFR amplification in its various forms have been developed. These meth- ods can be easily applied in many pathology laboratories. However, to determine the best way to test EGFR for adjusting treatment and whether the measurement of one form of EGFR is sufficient or more is needed, it must be compared to the clinical results of the new EGFR inhibitors. E-PS-15-047 Implementation of the 2021 WHO Classification of Tumours of the Central Nervous System and molecular assessment of diffuse gliomas in a Belgian cohort L.C. van Kempen*, M. Ahmed, A. Sieben, B. Verbraeken, T. Meno- vsky, M. Rasschaert, P. Meijnders, P. Cras, M. Lammens *Department of pathology, University of Antwerp, University Hospital Antwerp, Edegem, Belgium, Department of pathology and medical biology, University of Groningen, University Medical Center Gronin- gen, The Netherlands Background & objectives: The implementation of the 2021 WHO CNS tumour classification has introduced molecular analyses as an ancillary diagnostic test. The objective of this study was to investigate how often a molecular result contributed to the final diagnosis of dif- fuse glioma. Methods: All cases with a diagnosis of IDH1/2-wild type diffuse gli- oma were included (n=148). The time span for inclusion was limited to the period between November 1st, 2021 (the implementation of the 2021 WHO CNS our centre) until March 31st. Molecular testing on the DNA and RNA levels was a requirement for inclusion. Results: A TERT promotor mutation was observed in 117/148 cases (79%), a rate comparable with the literature. An isolated TERT mutation was observed in 12/148 (8%) of the cases. Morphological diffuse glioma grade 2 or 3 was upgraded to glioblastoma based on molecular alterations alone in 17/148 cases (i.e. molecular GBM, 11%). Sixteen of these 17 cases showed a TERT promotor mutation, 7 cases showed an EGFR amplification, and 6 cases showed both alterations. At the RNA level, different gene fusion transcripts were detected: EGFR, FGFR3, PTRRZ, MET and ROS1. Only one case of IDH1/2-wild type low-grade diffuse glioma did not contain a molecular alterations. Conclusion: Implementation of molecular diagnostics for CNS tumours according to the current WHO guideline resulted in upgrading of IDH-wild type diffuse glioma, (grade 2 or 3) to IDH- wild glioblastoma, (grade 4) in 11% of cases based on the molecu- lar results only. This has a considerable impact on the treatment trajectory for these patients. An isolated TERT promotor mutation was seen in 8% of all cases, which may also have prognostic conse- quences. Furthermore, molecular results pointed towards potential targeted therapy options. E-PS-15-049 Mitochondrial genome variants associated with pathogenesis of colorectal cancer O. Vasyukova*, N. Shakhpazyan, N. Sadykhov, V. Pechnikova, L. Mikhaleva *Avtsyn Research Institute of Human Morphology of Federal State Budgetary Scientific Institution "Petrovsky National Research Centre of Surgery", Russia Background & objectives: Mitochondria contribute to colorectal cancer (CRC) pathogenesis. Identified mitochondrial genome vari- ants (MGVs) are hypothesized to be associated with the inflammatory response and the innate immunity regulation in CRC. The study aimed to evaluate MGVs heteroplasmy changes in tumour tissue. Methods: We examined 12 patients (59-70 years old, 5 females, 7 males) with stage pT2T0N0 CRC (adenocarcinoma of the colon or sigmoid colon). Quantitative PCR was used to analyse heteroplasmy of MGVs m.652delG, m.1555A>G, m.3336T>C, m.3256C>T, m.5178C>A, m.12315G>A, m.13513G>A, m.14459G>A, m.14846G>A, and m.15059G>A in tumour and healthy colon tissue. Nonparametric statistical methods were employed for comparisons, using the U-test. Results: It was revealed that MGVs m.14459G>A and m.15059G>A in genes MT-ND6 and MT-CYTB, respectively, exhibited a higher level of heteroplasmy in tumour tissue compared to healthy tissue from the same patient (p<0.05). Other variants did not demonstrate significant increases or decreases in heteroplasmy levels in tissues. No significant associations were found between heteroplasmy levels and age, gender, or disease stage. Since the tumour tissue analysed comprised tumour cells and the microenvironment, including immune cells, the study results do not pinpoint the specific cells in the patho- logical tissue where MGV accumulation occurred. Nevertheless, the findings suggest that changes in MGV heteroplasmy may play a role in CRC pathogenesis. Conclusion: Observed differences in MGV heteroplasmy may be associated with altered cellular processes within tumour microenviron- ment, including cancer and immune cells. These changes could impact energy metabolism, apoptosis, or inflammation regulation, contribut- ing to CRC pathogenesis. The discovered MGVs might play a role in modulating tumour cell behaviour or interactions with the surrounding microenvironment. Further research is needed to elucidate the under- lying mechanisms, identify the specific cellular processes affected by MGV heteroplasmy differences, and determine their potential thera- peutic implications in colorectal cancer. Funding: Research was supported by RSF, grant № 23-25-00196