Ovarian cancer is the most lethal gynecologic malignancy, mainly due to late-stage diagnosis, frequent recurrences, and eventually therapy resistance. To identify potentially actionable genetic variants, sequencing data of 351 Belgian ovarian cancer patients were retrospectively captured from electronic health records. The cohort included 286 (81%) patients with high-grade serous ovarian cancer, 17 (5%) with low-grade serous ovarian cancer, and 48 (14%) with other histotypes. Firstly, an overview of the prevalence and spectrum of the BRCA1/2 variants highlighted germline variants in 4% (11/250) and somatic variants in 11% (37/348) of patients. Secondly, application of a multi-gene panel in 168 tumors revealed a total of 214 variants in 28 genes beyond BRCA1/2 with a median of 1 (IQR, 1–2) genetic variant per patient. The ten most often altered genes were (in descending order): TP53, BRCA1, PIK3CA, BRCA2, KRAS, ERBB2 (HER2), TERT promotor, RB1, PIK3R1 and PTEN. Of note, the genetic landscape vastly differed between the studied histotypes. Finally, using ESCAT the clinical evidence of utility for every genetic variant was scored. Only BRCA1/2 pathogenic variants were classified as tier-I. Nearly all patients (151/168; 90%) had an ESCAT tier-II variant, most frequently in TP53 (74%), PIK3CA (9%) and KRAS (7%). In conclusion, our findings imply that although only a small proportion of genetic variants currently have direct impact on ovarian cancer treatment decisions, other variants could help to identify novel (personalized) treatment options to address the poor prognosis of ovarian cancer, particularly in rare histotypes.

Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines and 3D tumor models. To address this goal, a well-characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G>T (p.(Gly12Val)) was used. Fluorescently labeled tumor cells were injected into the perivitelline space of 2 days’ post-fertilization zebrafish embryos. At 1 day post-injection, xenografts were assessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib and trametinib + luminespib. Subsequently, xenografts were euthanized and analyzed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses (n = 84) in vivo, with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.

Pancreatic cancer has a 5‐year survival rate of 12%, highlighting the need for reliable biomarkers for early detection and disease monitoring. Circulating tumor cells (CTCs) have emerged as a promising biomarker, yet their detection remains challenging. This study evaluates the Parsortix® system, a microfluidic device that enriches CTCs based on size and deformability, using pancreatic cancer cell lines. As increasing evidence indicates that during epithelial‐to‐mesenchymal transition (EMT) a cell's deformability increases, we evaluated possible biases by the device. The EMT stage of three pancreatic cancer cell lines, CAPAN‐1, MIA PaCa‐2, and PANC‐1, was assessed to classify them as epithelial, mesenchymal‐like, and hybrid, respectively. Spike‐in experiments showed that epithelial and hybrid phenotypes were more efficiently captured (62.6 ± 18.5% and 65.4 ± 11.1%) than mesenchymal‐like cancer cells (32.8 ± 10.2%). These results were confirmed using an EMT‐inducible breast cancer cell line. Lower recovery rates were found for the cells in a mesenchymal‐like state (31.5 ± 6.4%) than those in an epithelial state (47.56 ± 7.2%). In conclusion, the Parsortix® device may underestimate the number of mesenchymal CTCs.

Ovarian cancer (OC) is an umbrella term for cancerous malignancies affecting the ovaries, yet treatment options for all subtypes are predominantly derived from high-grade serous ovarian cancer, the largest subgroup. The concept of "functional precision medicine" involves gaining personalized insights on therapy choice, based on direct exposure of patient tissues to drugs. This especially holds promise for rare subtypes like low-grade serous ovarian cancer (LGSOC). This study aims to establish an in vivo model for LGSOC using zebrafish embryos, comparing treatment responses previously observed in mouse PDX models, cell lines, and 3D tumor models. To address this goal, a well characterized patient-derived LGSOC cell line with the KRAS mutation c.35 G > T (p.(Gly12Val)) was used. Fluorescently labelled tumor cells were injected into the perivitelline space of 2 days post fertilization zebrafish embryos. At 1 day post-injection xenografts were as-sessed for tumor size, followed by random allocation into treatment groups with trametinib, luminespib, and trametinib + luminespib. Subsequently xenografts were euthanized and analysed for apoptosis and proliferation by confocal microscopy. Tumor cells formed compact tumor masses (n=84) in vivo and with clear Ki67 staining, indicating proliferation. Zebrafish xenografts exhibited sensitivity to trametinib and luminespib, individually or combined, within a two-week period, establishing them as a rapid and complementary tool to existing in vitro and in vivo models for evaluating targeted therapies in LGSOC.

Pancreatic cancer has a 5-year survival rate of merely 12%. The high rate of late-stage diagnoses underscores the need for reliable biomarkers for early detection and disease monitoring. Circulating tumor cells (CTCs) have emerged as a promising biomarker, yet their detection remains challenging due to their rarity and phenotypic diversity. This study evaluates the Parsortix® system, a microfluidic device designed to enrich CTCs based on size and deformability, using pancreatic cancer cell lines. As increasing evidence indicates that during epithelial to mesenchymal transition (EMT) a cell’s deformability increases, we evaluated to what extent the Parsortix® system was biased towards epithelial cancer cells. First, the EMT stage of three pancreatic cancer cell lines, CAPAN-1, PANC-1 and MIA PaCa-2, was assessed using immunocytochemistry, flow cytometry and proteomics. CAPAN-1 cells were classified as epithelial, MIA PaCa-2 cells exhibited a mesenchymal-like phenotype, and PANC-1 cells demonstrated a hybrid phenotype. Then, by spiking these cells into blood samples, we determined the Parsortix® system’s ability to recover the cancer cells. Our results indicated that epithelial and hybrid phenotypes are more efficiently captured (62.6 ± 18.5% and 65.4 ± 11.1%) than mesenchymal cancer cells (32.8 ± 10.2%). To confirm these findings, spike-in experiments were repeated using an EMT inducible cell line. Again, significantly lower recovery rates were found for the cells in a mesenchymal-like state (31.5 ± 6.4%) compared to those in an epithelial state (47.56 ± 7.2%). In conclusion, the Parsortix® system may underestimate the presence of mesenchymal CTCs.