Validating reference-based algorithms to determine cell-type heterogeneity in ovarian cancer DNA methylation studies.

Information about cell composition in tissue samples is crucial for biomarker discovery and prognosis. Specifically, cancer tissue samples present challenges in deconvolution studies due to mutations and genetic rearrangements. Here, we optimized a robust, DNA methylation-based protocol, to be used for deconvolution of ovarian cancer samples. We compared several state-of-the-art methods (HEpiDISH, MethylCIBERSORT and ARIC) and validated the proposed protocol in an in-silico mixture and in an external dataset containing samples from ovarian cancer patients and controls. The deconvolution protocol we eventually implemented is based on MethylCIBERSORT. Comparing deconvolution methods, we paid close attention to the role of a reference panel. We postulate that a possibly high number of samples (in our case: 247) should be used when building a reference panel to ensure robustness and to compensate for biological and technical variation between samples. Subsequently, we tested the performance of the validated protocol in our own study cohort, consisting of 72 patients with malignant and benign ovarian disease as well as in five external cohorts. In conclusion, we refined and validated a reference-based algorithm to determine cell type composition of ovarian cancer tissue samples to be used in cancer biology studies in larger cohorts.

Potential Targeted Therapies in Ovarian Cancer.

BACKGROUND: We aimed to identify somatic pathogenic and likely pathogenic mutations using next-generation sequencing (NGS). The mutational findings were held against clinically well-described data to identify potential targeted therapies in Danish patients diagnosed with high-grade serous ovarian cancer (HGSC). METHODS: We characterized the mutational profile of 128 HGSC patients. Clinical data were obtained from the Danish Gynecological Database and tissue samples were collected through the Danish CancerBiobank. DNA was analyzed using NGS. RESULTS: 47 (37%) patients were platinum-sensitive, 32 (25%) partially platinum-sensitive, 35 (27%) platinum-resistant, and three (2%) platinum-refractory, while 11 (9%) patients did not receive chemotherapy. Overall, 27 (21%) had known druggable targets. Twelve (26%) platinum-sensitive patients had druggable targets for PARP inhibitors: one for tyrosine kinase inhibitors and one for immunotherapy treatment. Eight (25%) partially platinum-sensitive patients had druggable targets: seven were eligible for PARP inhibitors and one was potentially eligible for alpesilib and hormone therapy. Seven (20%) platinum-resistant patients had druggable targets: six (86%) were potentially eligible for PARP inhibitors, one for immunotherapy, and one for erdafitinib. CONCLUSIONS: PARP inhibitors are the most frequent potential targeted therapy in HGSC. However, other targeted therapies remain relevant for investigation according to our mutational findings.

The AMSH3 ESCRT-III-Associated Deubiquitinase Is Essential for Plant Immunity.

Plant "nucleotide-binding leucine-rich repeat" receptor proteins (NLRs) detect alterations in host targets of pathogen effectors and trigger immune responses. The Arabidopsis thaliana mutant pen1 syp122 displays autoimmunity, and a mutant screen identified the deubiquitinase "associated molecule with the SH3 domain of STAM3" (AMSH3) to be required for this phenotype. AMSH3 has previously been implicated in ESCRT-mediated vacuolar targeting. Pathology experiments show that AMSH3 activity is required for immunity mediated by the CC-NLRs, RPS2 and RPM1. Co-expressing the autoactive RPM1D505V and the catalytically inactive ESCRT-III protein SKD1E232Q in Nicotiana benthamiana supports the requirement of ESCRT-associated functions for this CC-NLR-activated immunity. Meanwhile, loss of ESCRT function in A. thaliana is lethal, and we find that AMSH3 knockout-triggered seedling lethality is "enhanced disease susceptibility 1" (EDS1) dependent. Future studies may reveal whether AMSH3 is monitored by a TIR-NLR immunity receptor.