10P an Adapted CGP-based Model to Interpret POLE Mutations in Endometrial Cancer

Endometrial cancer (EC) patients harbouring POLE mutations have been shown exceptionally good survival outcomes allowing de-escalation of adjuvant treatment. POLE encodes for the Pol ε catalytic subunit capable of correcting DNA synthesis errors; pathogenic mutations within the exonuclease domain (EDM) result in an ultramutated phenotype. So far, 11 POLE EDM variants have been recognized as hotspot mutations. Given the increasing availability in clinical practice of comprehensive genome profiling (CGP), the interpretation of non-hotspot EDM variants has become challenging. EC patients enrolled in a CGP programme at Fondazione Policlinico Universitario Agostino Gemelli IRCCS were profiled using TSO500HT. VCF files containing identified variants were analyzed developing an in-house Python and R language script. The following parameters including the frequency of C > A, C > G, T > G substitutions, tumor mutational burden (TMB), the frequency of indels, and the presence of recurrent mutations were evaluated and a score to interpret POLE mutations was recalibrated from Castillo et al. published score on TCGA data. From January 2022 to May 2023, CGP was available for 392 EC patients. New thresholds for the frequency of C > A, C > G, T > G substitutions and indels were calculated based on the mean of the median values of each parameter comparing POLE EDM hotspots vs POLE benign variants and wild type (WT) cases. Overall, the score correctly identified the POLE hotspot mutations in 40/42 patients. One of the 2 misclassified cases was a FIGO stage IIIC1 case, displaying a TMB of 36.5; the other displayed a TMB of 87. 5 POLE EDM non-hotspot variants were identified (D287E, N363K, T278M, D262H, R375Q) and none of them reached a score ≥ 4. None of these cases displayed a COSMIC signature 10 as expected for POLE EDM hotspot cases. Among the co-mutations, ATM and APC variants were reported to be differentially expressed among POLE EDM hotspots and POLE EDM non-hotspots vs POLE benign and WT cases (ATM: 60%, 60% vs 10%, 11%; APC: 35%, 20% vs 3.8% and 0% respectively). We adapted Castillo’s TCGA-based POLE score to a CGP assay. This approach could be helpful to interpret POLE EDM non hotspots variants emerging from wide routine genomic characterization.