Caroline van den Berg

Epigenetic and Genomic Hallmarks of PARP-Inhibitor Resistance in Ovarian Cancer Patients

Background: Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance. Materials and Methods: Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures. Results: Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ2-test p < 0.001). Conclusion: Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.

Genome-wide cell-free DNA methylation profiling in advanced stage ovarian cancer. Are we looking at the tumor or the patient's immune response to the tumor?

The aim of this study was to identify differentially methylated regions in cell-free DNA (cfDNA) between healthy persons and patients with advanced stage ovarian cancer (ASOC) and to identify differences in cfDNA methylation before and after cytoreductive surgery. Plasma-derived cfDNA was analyzed by a high-throughput genome wide DNA methylation sequencing technique: MeD-seq. A training set of therapy naïve cfDNA samples of patients with ASOC (≥FIGO stage IIIB, n=10) was compared with cfDNA of healthy controls (n=10) to define a ASOC specific cfDNA methylation signature. A cumulative hypermethylation score was constructed and a validation set of pre- and postoperative samples of 39 patients were compared using this score. MeD-seq results of tumor tissue samples were correlated with cfDNA results. Patients with ASOC showed a clear distinct cfDNA methylation signature from healthy controls (p<0.0001). This cfDNA-methylation signature resulted in preoperative hypermethylation scores (135; interquartile range 110-163) that were significantly higher than postoperative hypermethylation scores (91; interquartile range 76-101) (p<0.001). The cfDNA methylation signature at baseline differed from tumor tissue and was more closely related to DNA methylation of immune-related cells (T-lymphocytes, neutrophil granulocytes, monocytes, and B-lymphocytes) than to ASOC tissue. MeD-seq provides a promising method for genome wide methylation profiling of cfDNA. Patients with ASOC could clearly be distinguished from healthy controls and differed pre- and postoperatively.

Poor accuracy of endometrial sampling in patients with uterine carcinosarcomas: a nationwide analysis

To determine the accuracy of aspiration biopsy (AB), hysteroscopic biopsy (HB), and dilatation & curettage (D&C) in detecting uterine carcinosarcoma (UCS). Pathology reports were retrieved from the Dutch Nationwide Pathology Databank PALGA for patients with a certain or suggested diagnosis of UCS in pre- and/or postoperative histology between 2001 and 2021. Patients without available pre- or postoperative pathology reports were excluded. The accuracy measures sensitivity, positive predictive value (PPV), accuracy, and concordance using Cohen's kappa were calculated for AB, D&C, and HB, using postoperative histology as the reference. This was analyzed for 2 scenarios: Analysis A compared samples with a certain or suggested diagnosis of UCS vs. no mention of UCS. Analysis B compared samples with a certain diagnosis of UCS vs those without UCS. The study included 1,481 patients, totaling 1,685 samples. Sensitivity was similar for AB and HB (52.4% and 50.5%, respectively, for analysis A; 45.1% and 42.2% for analysis B). D&C showed the highest sensitivity (70.8% and 64.9% for analysis A and B, respectively). AB had the highest PPV (85.3% and 90.9% for analysis A and B, respectively), HB had the lowest PPV (79.7% and 80.9%, respectively). Accuracy was highest for D&C (44.4%) compared to AB (32.8%) and HB (29.5%). All Cohen's kappa values were below 0.20, indicating poor correlation between preoperative and postoperative diagnoses. The study reveals low accuracy measures across all conventional endometrial sampling techniques, highlighting the need for research to identify markers or tools to diagnose UCS.