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Purpose: High-grade serous epithelial ovarian cancer (HGS-EOC) is defined by high levels of somatic copy-number alterations (SCNA) with marked spatial and temporal tumor heterogeneity. Biomarkers serving to monitor drug response and detect disease recurrence are lacking, a fact which reflects an unmet clinical need. Experimental Design: A total of 185 plasma samples and 109 matched tumor biopsies were collected from 46 patients with HGS-EOC, and analyzed by shallow whole-genome sequencing (sWGS). The percentage of tumor fraction (TF) in the plasma was used to study the biological features of the disease at the time of diagnosis (T0) and correlated with patients' survival. Longitudinal analysis of TF was correlated with CA-125 levels and radiological images to monitor disease recurrence. Results: Gain in the clonal regions, 3q26.2 and 8q24.3, was observed in the 87.8% and 78.05% of plasma samples, suggesting that plasmas WGS mirrors solid biopsies. AtT0, multivariate analysis revealed that plasma TF levels were an independent prognostic marker of relapse (P < 0.022). After platinum (Pt)-based treatment, circulating tumor DNA (ctDNA) analysis showed a change in the heterogeneous pattern of genomic amplification, including an increased frequency of amplification, compared with before Ptbased treatment in the 19p31.11 and 19q13.42 regions. TF in serially collected ctDNA samples outperformed CA-125 in anticipating clinical and radiological progression by 240 days (range, 37-491). Conclusions: Our results support the notion that sWGS is an inexpensive and useful tool for the genomic analysis of ctDNA in patients with HGS-EOC to monitor disease evolution and to anticipate relapse better than serum CA-125, the routinely used clinical biomarker.

High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches.

Stage I epithelial ovarian cancer (EOC) represents about 10% of all EOCs. It is characterized by a complex histopathological and molecular heterogeneity, and it is composed of five main histological subtypes (mucinous, endometrioid, clear cell and high, and low grade serous), which have peculiar genetic, molecular, and clinical characteristics. As it occurs less frequently than advanced-stage EOC, its molecular features have not been thoroughly investigated. In this study, using in silico approaches and gene expression data, on a multicentric cohort composed of 208 snap-frozen tumor biopsies, we explored the subtype-specific molecular alterations that regulate tumor aggressiveness in stage I EOC. We found that single genes rather than pathways are responsible for histotype specificities and that a cAMP-PKA-CREB1 signaling axis seems to play a central role in histotype differentiation. Moreover, our results indicate that immune response seems to be, at least in part, involved in histotype differences, as a higher immune-reactive behavior of serous and mucinous samples was observed with respect to other histotypes.

OBJECTIVES:To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. DESIGN:Prospective experimental study. SETTING:Laboratory research. SUBJECTS:C57Bl/6 mice. INTERVENTIONS:Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. MEASUREMENTS AND MAIN RESULTS:Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. CONCLUSIONS:Human amniotic mesenchymal stromal cell-secreted factors protect

High grade serous epithelial ovarian cancer (HGS-EOC) is the most lethal gynaecological disease, usually diagnosed at advanced stages. Although most HGS-EOC patients are initially sensitive to therapy, about 20% of them does not respond to front-line platinum (Pt)-based chemotherapy, relapsing within 6 months from treatment. No molecular biomarkers are as yet available in the clinical practice to discriminate, at the time of diagnosis, the patients who will respond or not to first-line Pt treatment. The identification of a molecular signature associated with intrinsic Pt resistance would allow to select patients who will not respond to Pt compound, thus avoiding ineffective treatment and related toxicities. In this thesis, defects in transcriptional regulation mechanisms associated to intrinsic Pt resistance were investigated using two different omic approaches – microarray and RNA sequencing. Moreover, considering the correlation between defects in DNA repair Homologous Recombination and Pt sensitivity, we have started to develop an academic assay which exploits high-throughput DNA sequencing to evaluate Homologous Recombination Deficiency (HRD). Results obtained from three independent HGS-EOC cohorts (n=1080) indicate that the combined expression profile of three genes – PRKG1, SDF2L1 and PPP1R12A – could represent an independent prognostic biomarker for Pt response and survival. In addition, 25 transcripts, 16 of which are unknown or partially known variants, have been identified that are differentially expressed between Pt-sensitive and Pt-resistant patients. These results, once validated, integrated and associated with the HRD status, could identify a molecular signature applicable in the clinical setting to enable stratification of patients at time of diagnosis, allowing to choose the best therapeutic option.

High grade serous epithelial ovarian cancer (HGS-EOC) is the most lethal gynaecological disease, usually diagnosed at advanced stages. Although most HGS-EOC patients are initially sensitive to therapy, about 20% of them does not respond to front-line platinum (Pt)-based chemotherapy, relapsing within 6 months from treatment. No molecular biomarkers are as yet available in the clinical practice to discriminate, at the time of diagnosis, the patients who will respond or not to first-line Pt treatment. The identification of a molecular signature associated with intrinsic Pt resistance would allow to select patients who will not respond to Pt compound, thus avoiding ineffective treatment and related toxicities. In this thesis, defects in transcriptional regulation mechanisms associated to intrinsic Pt resistance were investigated using two different omic approaches – microarray and RNA sequencing. Moreover, considering the correlation between defects in DNA repair Homologous Recombination and Pt sensitivity, we have started to develop an academic assay which exploits high-throughput DNA sequencing to evaluate Homologous Recombination Deficiency (HRD). Results obtained from three independent HGS-EOC cohorts (n=1080) indicate that the combined expression profile of three genes – PRKG1, SDF2L1 and PPP1R12A – could represent an independent prognostic biomarker for Pt response and survival. In addition, 25 transcripts, 16 of which are unknown or partially known variants, have been identified that are differentially expressed between Pt-sensitive and Pt-resistant patients. These results, once validated, integrated and associated with the HRD status, could identify a molecular signature applicable in the clinical setting to enable stratification of patients at time of diagnosis, allowing to choose the best therapeutic option.