Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 ß (HERß-1)-pathway, with HERß-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Nuclear receptor co-repressor NCOR2 and its relation to GPER with prognostic impact in ovarian cancer.

PURPOSE: The significance of the non-classical G-protein-coupled estrogen receptor (GPER) as positive or negative prognostic factor for ovarian cancer patients remains still controversial. Recent results indicate that an imbalance of both co-factors and co-repressors of nuclear receptors regulates ovarian carcinogenesis by altering the transcriptional activity through chromatin remodeling. The present study aims to investigate whether the expression of the nuclear co-repressor NCOR2 plays a role in GPER signaling which thereby could positively impact overall survival rates of ovarian cancer patients. METHODS: NCOR2 expression was evaluated by immunohistochemistry in a cohort of 156 epithelial ovarian cancer (EOC) tumor samples and correlated with GPER expression. The correlation and differences in clinical and histopathological variables as well as their effect on prognosis were analyzed by Spearman's correlation, Kruskal-Wallis test and Kaplan-Meier estimates. RESULTS: Histologic subtypes were associated with different NCOR2 expression patterns. More specifically, serous and mucinous EOC demonstrated a higher NCOR2 expression (P = 0.008). In addition, high nuclear NCOR2 expression correlated significantly with high GPER expression (cc = 0.245, P = 0.008). A combined evaluation of both high NCOR2 (IRS > 6) and high GPER (IRS > 8) expression revealed an association of a significantly improved overall survival (median OS 50.9 versus 105.1 months, P = 0.048). CONCLUSION: Our results support the hypothesis that nuclear co-repressors such as NCOR2 may influence the transcription of target genes in EOC such as GPER. Understanding the role of nuclear co-repressors on signaling pathways will allow a better understanding of the factors involved in prognosis and clinical outcome of EOC patients.

Diagnostic workup for endometrioid borderline ovarian tumors (eBOT) requires histopathological evaluation of the uterus.

BACKGROUND: For young borderline ovarian tumor (BOT) patients, preservation of the uterus was incorporated as an accepted option into treatment guidelines. For the endometrioid subtype (eBOT) however, adequate histological evaluation is challenging and might be associated with synchronous endometrial disorders or misinterpreted as spread from uterine primaries. CASE PRESENTATION: We report the cases of two young patients with eBOT who underwent treatment according to current guidelines. In both cases, unexpected findings of invasive uterine carcinomas were established in final histopathological evaluation. CONCLUSIONS: This constellation highlights the challenging diagnostic workup of BOT and underlines that uterine curettage is indispensable for eBOT to exclude uterine primary tumors when fertility preservation is planned. Accordingly, we suggest to include this procedure into recommendations for diagnostic workup and to state the potential risk in treatment guidelines.

Paternal age effects on sperm FOXK1 and KCNA7 methylation and transmission into the next generation.

Children of older fathers carry an increased risk for developing autism and other disorders. To elucidate the underlying mechanisms, we investigated the correlation of sperm DNA methylation with paternal age and its impact on the epigenome of the offspring. Methylation levels of nine candidate genes and LINE-1 repeats were quantified by bisulfite pyrosequencing in sperm DNA of 162 donors and 191 cord blood samples of resulting children (conceived by IVF/ICSI with the same sperm samples). Four genes showed a significant negative correlation between sperm methylation and paternal age. For FOXK1 and KCNA7, the age effect on the sperm epigenome was replicated in an independent cohort of 188 sperm samples. For FOXK1, paternal age also significantly correlated with foetal cord blood (FCB) methylation. Deep bisulfite sequencing and allele-specific pyrosequencing allowed us to distinguish between maternal and paternal alleles in FCB samples with an informative SNP. FCB methylation of the paternal FOXK1 allele was negatively correlated with paternal age, whereas maternal allele was unaffected by maternal age. Since FOXK1 duplication has been associated with autism, we studied blood FOXK1 methylation in 74 children with autism and 41 age-matched controls. The FOXK1 promoter showed a trend for accelerated demethylation in the autism group. Dual luciferase reporter assay revealed that FOXK1 methylation influences gene expression. Collectively, our study demonstrates that age-related DNA methylation changes in sperm can be transmitted to the next generation and may contribute to the increased disease risk in offspring of older fathers.

Human performance consequences of automated decision aids in states of sleep loss.

OBJECTIVE: The authors investigated how human performance consequences of automated decision aids are affected by the degree of automation and the operator's functional state. BACKGROUND: As research has shown, decision aids may not only improve performance but also lead to new sorts of risks.Whereas knowledge exists about the impact of system characteristics (e.g., reliability) on human performance, little is known about how these performance consequences are moderated by the functional state of operators. METHOD: Participants performed a simulated supervisory process control task with one of two decision aids providing support for fault identification and management. One session took place during the day, and another one took place during the night after a prolonged waking phase of more than 20 hr. RESULTS: Results showed that decision aids can support humans effectively in maintaining high levels of performance, even in states of sleep loss, with more highly automated aids being more effective than less automated ones. Furthermore, participants suffering from sleep loss were found to be more careful in interaction with the aids, that is, less prone to effects of complacency and automation bias. However, cost effects arose that included a decline in secondary-task performance and an increased risk of return-to-manual performance decrements. CONCLUSION: Automation support can help protect performance after a period of extended wakefulness. In addition, operators suffering from sleep loss seem to compensate for their impaired functional state by reallocating resources and showing a more attentive behavior toward possible automation failures. APPLICATION: Results of this research can inform the design of automation, especially decision aids.