e15059 Background: Non–small cell lung cancer (NSCLC) remains associated with poor prognosis in advanced stages, and reliable biomarkers for monitoring treatment response are still limited. While liquid biopsy applications have largely focused on circulating nucleic acids, extracellular vesicles (EVs) represent a stable and biologically informative source of tumor- and microenvironment-derived proteins. EV proteomic profiling may provide dynamic insights into therapy-induced molecular changes and improve patient stratification. Methods: Plasma samples were collected from patients with advanced NSCLC at baseline (P0, pre-treatment) and at first radiological evaluation (P1), prior informed consent was obtained. Patients were stratified according to treatment regimen (chemotherapy, immunotherapy, or chemo-immunotherapy). EVs were isolated from plasma using an affinity-based enrichment method optimized for low sample volumes and characterized by nanoparticle tracking analysis and Western blotting. EV protein cargo was analyzed by data-independent acquisition (DIA) LC–MS/MS with label-free quantification. Multivariate analyses, functional enrichment (GO/KEGG), and protein–protein interaction network analyses were performed to identify treatment- and time-dependent proteomic signatures. Results: EV proteomic profiling identified 418 EV-associated proteins across all samples. Comparison between baseline (P0) and post-treatment (P1) revealed 108 differentially expressed proteins (43 enriched at P0, 65 at P1). In immunotherapy-treated patients, baseline EVs displayed heterogeneous profiles enriched in epithelial organization, lipid metabolism, complement activation, and coagulation pathways. Post-treatment EVs exhibited marked proteomic remodeling, with enrichment of immune modulation, oxidative stress response, metabolic adaptation, and cytoskeletal remodeling pathways. Functional analyses indicated increased complement regulation, protease inhibition, and immune–stromal interactions, consistent with therapy-induced tumor microenvironment reprogramming. Post-treatment EV profiles partially converged with other treatment groups while retaining immunotherapy-specific signatures. Conclusions: These findings support further study of plasma-derived EVs as they capture dynamic proteomic changes associated with immune checkpoint inhibition in NSCLC and represent a promising non-invasive tool for monitoring immunotherapy-induced molecular remodeling and for biomarker-driven patient stratification and follow-up.

e17050 Background: 177Lu-PSMA-617 radioligand therapy (RLT) has demonstrated efficacy in metastatic castration-resistant prostate cancer (mCRPC), although clinical benefit remains heterogeneous. We hypothesize that loss of PTEN leads to the functional impairment of FANCD2 -mediated high-fidelity DNA damage repair (DDR). In this deficient landscape, mCRPC cells become hyper-dependent on POLQ , which governs the error-prone Microhomology-Mediated End Joining (MMEJ) pathway. This "pathway switching" may sensitize mCRPC cells to RLT-induced DDR. We investigated whether baseline clinical factors and cell-free nucleic acid (cfNA) molecular profiles could refine patient selection and predict biochemical response. Methods: Consecutive mCRPC, PSMA-positive, patients treated with 177Lu-PSMA-617 every 6 weeks, from May to October 2025, were included. Baseline clinical data [Body Mass Index (BMI) Neutrophil-to-Lymphocyte Ratio (NLR), NRS pain scale, and time to CRPC)] were collected. Liquid biopsies were obtained after 3 cycles. Gene expression of PTEN , POLQ , and FANCD2 was quantified via ddPCR from plasma-derived cfNA. Biochemical response (BR) was defined as a ≥50% PSA decline. Results: Among 31 evaluable patients, 16 were responders (R) and 15 non-responders (NR). R showed lower baseline BMI (25.2 vs 26.8 months), NLR (2.6 vs 3.5), and NRS (5 vs 7), with a significantly longer time to CRPC (23 vs 13). Liquid biopsy showed lower median cfDNA levels in R (0.35 vs 1.57 ng/µL). Notably, a distinct DDR signature was associated with BR: R exhibited significantly lower median expression of PTEN (11.74 vs 14.83; p = 0.005) and FANCD2 (2.99 vs 13.65; p = 0.03), while POLQ expression was markedly elevated or uniquely detectable in R (range 0–27.23) compared to NR (0–0.83). This molecular signature suggests that the downregulation of PTEN and FANCD2 induces a homologous recombination deficiency state, providing a compensatory activation of the error-prone POLQ -mediated MMEJ pathway. Conclusions: A cfNA profile characterized by reduced FANCD2/PTEN and increased POLQ expression identifies a "synthetic vulnerability" signature of high genomic instability where the loss of high-fidelity repair mechanisms renders the tumor highly susceptible to RLT. These findings support futher study of PTEN-FANCD2-POLQ axis as predictive biomarker in larger cohorts, and the development of combination strategies targeting the DDR axis.

e22507 Background: Currently,Breast Cancer (BC) is not considered a mismatch repair (MMR)-deficiency (MMRd)–spectrum tumor. The BC patients carrying constitutional MMR pathogenic variants (PVs) are not eligible for dedicated screening programs and risk-reducing strategies, which are addressed only to the BC individual carriers of specific Homologous Recombination Repair (HRR) variants. The MMRd status, as well as the high levels of microsatellite instability (MSI-H) that occur upon loss of the expression of MMR genes, may have direct therapeutic implications for the patients, who may benefit from the treatment with immune-checkpoint inhibitors (ICIs). Methods: This was an observational, hospital-based, cohort study to investigate the prevalence of germline MMR PVs in BC patients who were appropriate candidates for hereditary breast and/or ovarian cancer (HBOC) predisposition evaluation. BC patients were tested through NGS-based multigene panel testing including MMR-associated genes, according to clinical established criteria. Results: Between January 2016 and December 2024, 6.020 BC patients were referred for genetic counseling and HBOC predisposition evaluation. In the cohort of consecutive 5.595 BC patients tested with multigene panel testing, a subgroup of 26 patients resulted carriers of germline MMR PVs. Interestingly, 6 of 26 MMR-mutated patients (23.1%) had also a personal bilateral BC history. The median age at BC diagnosis was 55 (range 31-82). Mutation prevalence showed that PMS2 was the most mutated MMR gene (10 PVs, 38.5%), followed by MLH1 (6 PVs, 23.1%), EPCAM (5 PVs, 19.2%), MSH2 (3 PVs, 11.5%), and MSH6 (2 PVs, 7.7%). Notably, in the MMR - mutated cohort, 73.1% of BC patients (19/26) reported a family history of HRR-associated tumors (BC, ovarian/prostate and/or pancreatic tumors), predominantly BC (78.9%). Conversely, first or second-degree family members with a colorectal or endometrial cancer diagnosis, that represents the main Lynch Syndrome-associate tumors, were reported only for 3 patients (11.5%). The genetic characteristics and the clinical behavior of BC in MMR-, BRCA - and HRR non- BRCA -carriers were also collected. The variant of uncertain significance (VUS) prevalence was described. Conclusions: Our results suggested that, in women who met hereditary BC testing criteria but missed the established Lynch Syndrome clinical or testing criteria, the use of panel testing including MMR genes could lead to the identification of a subgroup of BC patients who are carriers of MMR constitutional PVs, with potential important preventive and therapeutic implications.

Extracellular vesicle (EV) monitoring can complement clinical assessment of cancer response. In this study, patients with advanced non‐small cell lung cancer (NSCLC) undergoing osimertinib, alectinib, pembrolizumab or platinum‐based chemotherapy ± pembrolizumab were enrolled. EVs were characterized using Bradford assay to quantify the circulating cell‐free EV protein content (cfEV), and dynamic light scattering to assess Rayleigh ratio excess at 90°, z‐averaged hydrodynamic diameter and polydispersity index. A total of 135 plasma samples from 27 patients were collected at baseline (T0) and at the first radiological restaging (T1). A ∆cfEV < 20% was associated with improved median progression‐free survival (mPFS) in responders versus non‐responders. Specifically, cfEV responders on pembrolizumab had a significantly better mPFS (25.2 months) compared to those on chemotherapy plus pembrolizumab (6.1 months). EGFR‐positive cfEV responders also experienced longer mPFS compared to cfEV non‐responders (35.1 months, 95% CI: 14.9–35.5 vs. 20.8 months, 95% CI: 11.2–30.4). This study suggested that monitoring circulating EV could provide valuable insights into treatment efficacy in NSCLC, particularly for patients receiving pembrolizumab or osimertinib.

e22516 Background: Several case-control studies have been performed to understand the prognostic impact of pregnancy after breast cancer in patients carrying germline BRCA1/2 (g BRCA1/2 ) pathogenic/likely pathogenic variants (PVs). The median age of onset of epithelial ovarian cancer (EOC) diagnosis is higher than breast cancer. Clinical data on fertility, pregnancy outcomes, and abortion rates before the EOC onset are still missing. Previous research showed that g BRCA PV carriers may have a reduced ovarian reserve, earlier menopause than the general population, and defective homologous recombination repair of DNA double-strand breaks in human trophoblast, potentially leading to spontaneous miscarriage. Methods: This was a real-world, hospital-based cohort study to assess the cumulative incidence of pregnancy and miscarriage in a consecutive series of EOC patients who underwent g BRCA1/2 between May 2015 and December 2024. EOC patients carrying PVs in no- BRCA1/2 genes or tumor BRCA1/2 PVs were excluded from the current analysis. Results: From May 2015 to December 2024, 731 EOC patients were included in the analysis: 138 were carriers of g BRCA1/2 PVs (18.9%): 93 in BRCA1 (67.4%), and 45 in BRCA2 (32.6%). The number of patients with at least 1 pregnancy, abortion included, was 91 in the g BRCA carriers vs 192 in the g BRCA non-carriers. Notably, when the incidence of miscarriage between BRCA carriers and non-carriers was compared, the reduced incidence of spontaneous miscarriage was in the group of patients carrying g BRCA PVs [ BRCA carriers vs non-carriers: 11/92 (12.1%) vs 51/192 (26.6%)]. The total number of full-term pregnancies was 205 for g BRCA carriers vs 192 for non-carrier patients. Also, the median number of full-term pregnancies for single patients was higher in the patients harboring g BRCA PVs vs BRCA wild type (2.25 vs 1.5). The total number of miscarriages was 20 among g BRCA carriers vs 70 among non-carriers, with a lower miscarriage rate for individual patients in the BRCA -mutated patients (1.8 vs 1.3). The genetic characteristics and the EOC clinical behavior between parous and nulliparous women, according to BRCA mutational status, were also collected. Conclusions: Genetic diagnosis of constitutional BRCA1/2 PVs mutation raises concerns about the future fertility of female carriers. Interestingly, our data suggested that pregnancy in EOC women carriers of g BRCA1/2 PVs was associated with a reduced incidence of miscarriage than non-carriers. Prospective validation of these findings is required to improve understanding of fetal and obstetric outcomes and to guide counseling for BRCA carrier patients.

10543 Background: Homologous recombination repair (HRR) defects are driver mutational imprints and actionable biomarkers in prostate cancer (PrC) patients. However, recent research provides evidence of PrC onset also in the context of mismatch repair deficiency (MMRd). In the current report, we investigated whether the position of pathogenic variants (PVs) in the functional domains (FDs) of HRR genes could be associated with reduced or increased sensitivity to PARP-inhibitors (PARPis). We also questioned whether the HRR FDs had a prognostic significance and affected the development of second primary tumor (SPT) in males with a genetic predisposition. Methods: This was a real-world, observational, study including PrC patients undergoing germline, somatic, and liquid biopsy testing between January 2020 and December 2023. Prognostic factors, SPT history, and PARPi benefit were evaluated according to mutated genes ( BRCA1, BRCA2,No- BRCAHRR), PV location within the FDs, and germline (g) or somatic (s) PV. The BRCA1FDs were RING, DNA-binding domain (DBD), BRCA1 C terminus (BRCT), or other location, and the BRCA2 FDs were RAD51-BD, DBD, or other location. Results: A total of 833 metastatic PrC patients, aging 40 to 91, were included in this study; 169 (20.3%) were carriers of germline (n.48, 5.8%) or somatic (n.121, 14.5%) PVs in HRR genes: 17 in BRCA1 (g BRCA1: n.4, 2.4%; s BRCA1: n.13, 7.7% ), 102 in BRCA2 (g BRCA2: n.33, 19.5%; s BRCA2: n.69, 40.8% ), and 53 in no- BRCA HRR genes (gHRR: n.11, 6.5%; sHRR: n.42, 24.8%). CHEK2was the HRR gene most frequently affected by germline PVs (27.3%), while the most frequent somatic PVs were in the ATM (40.5%). Liquid biopsy identified PVs in 33 patients out of 185 tested, including n.10 (5.4%) not detected through somatic and germline testing, considerably expanding the therapeutic window for the use of PARPi. Notably, differences in PFS rates at 48 months were observed when comparing sub-groups according to PV location in the FDs of BRCA2.Surprisingly, when we investigated the SPT in the cohorts of patients with and without germline PVs, the difference was not significant (14.5% vs 12.4%, respectively) and, in the subgroup without germline PVs, the sites of SPT were predominantly colorectal, gastric, bladder or other tumors classically dominated by MMRd and recognized as Lynch Syndrome-spectrum tumors. This observation may result in unexplored forms of hypermutable tumor phenotypes, potentially impacting cancer risk management and increasing therapeutic opportunities. Conclusions: Our results suggest preliminary evidence that not all BRCA2-mutated PrC are equally sensitive to PARPis, and the response could depend on the PV location within FDs. The study of SPT in patients with no-HRR-associated PrC highlights the need to expand the genes in multigene panel testing, including MMR along with HRR genes, to capture unrecognized constitutional predisposition.

This preclinical proof-of-concept study aimed to evaluate the effectiveness of secretome therapy in diabetic mice with pressure ulcers. We utilized a custom-made hyaluronic acid (HA)-based porous sponge, which was rehydrated either with normal culture medium or secretome derived from human mesenchymal stromal cells (MSCs) to achieve a hydrogel consistency. Following application onto skin ulcers, both the hydrogel-only and the hydrogel + secretome combination accelerated wound closure compared to the vehicle group. Notably, the presence of secretome significantly enhanced the healing effect of the hydrogel, as evidenced by a thicker epidermis and increased revascularization of the healed area compared to the vehicle group. Notably, molecular analysis of healed skin revealed significant downregulation of genes involved in delayed wound healing and abnormal inflammatory response in ulcers treated with the hydrogel + secretome combination, compared to those treated with the hydrogel only. Additionally, we found no significant differences in therapeutic outcomes when comparing the use of secretome from fetal dermal MSCs to that from umbilical cord MSCs. This observation is supported by the proteomic profile of the two secretomes, which suggests a shared molecular signature responsible of the observed therapeutic effects.

Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

While platinum(ii)-based drugs continue to be employed in cancer treatments, the escalating occurrence of severe side effects has spurred researchers to explore novel sources for potential therapeutic agents. Notably, cobalt(iii) has emerged as a subject of considerable interest due to its ubiquitous role in human physiology. Several studies investigating the anticancer effects of Salphen complexes derived from cobalt(iii) have unveiled intriguing antiproliferative properties. In a bid to enhance our understanding of this class of compounds, we synthesized and characterized two novel half Salphen cobalt(iii) complexes. Both compounds exhibited notable stability, even in the presence of physiologically relevant concentrations of glutathione. The application of spectroscopic and computational methodologies unravelled their interactions with duplex and G4-DNAs, suggesting an external binding affinity for these structures, with preliminary indications of selectivity trends. Importantly, antiproliferative assays conducted on 3D cultured SW-1353 cancer cells unveiled a compelling anticancer activity at low micromolar concentrations, underscoring the potential therapeutic efficacy of this novel class of cobalt(iii) complexes.

e20036 Background: Lung cancerstill represents one of the most common and lethal diseases worldwide. There remains an unmet clinical need for the discovery of biomarkers to refine the management of patients with advanced non-small cell lung cancer (NSCLC). The combined evaluation of circulating cell-free DNA (cfDNA) and deep learning-based radiomic methods remains to be validated in the clinical setting. Methods: The RATIONALE study is a retro-prospective observational clinical trial consecutively enrolling treatment-naïve patients with advanced NSCLC who received standard first-line systemic treatments at Paolo Giaccone University Hospital, Palermo (Italy). Baseline and follow-up blood samples were collected according to computed tomography (CT) examinations performed using the same equipment (Siemens SOMATOM 128 slices), the same acquisition protocol, and the same contrast medium (Iomeron 400) every twelve weeks. All primary lung tumor internal regions of interest (ROIs) were segmented. Radiomic feature extraction was performed on the ROIs using Pyradiomics, an extension of the 3DSlicer. Circulating cfDNA was extracted from 1 ml of plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen) and quantified in terms of ng/μL using a QubitTM dsDNA HS Assay Kit (ThermoFisher Scientific). The diagnostic performance was evaluated using receiver operating characteristic (ROC) curves. Results: Between June 2021 and November 2023, fifty-five patients were included. Here we present the first results from the cohort of twenty patients undergoing first-line pembrolizumab-based treatments. Among the thirteen features extracted, the feature 'original_glcm_Id' evaluating the local image homogeneity emerged as statistically significant for differentiating adenocarcinoma and squamous cell carcinoma (sensitivity of 83.7%, specificity of 81.0%, accuracy of 82.8%; p < 0.001). When correlating cfDNA dynamics and radiologic response, out of the eight features examined, the feature 'first order_mean' measuring the average intensity of gray levels proved to be statistically significant (sensitivity of 86.0%, specificity of 87.7%, accuracy of 86.9%; p < 0.006). Finally, the feature 'size zone non-uniformity' measuring the variability of volumes in different gray level zones within the image showed a good performance in predicting overall survival (sensitivity of 65.8%, specificity of 100.0%, accuracy of 77.8%; p < 0.001). Conclusions: The preliminary results of this ongoing study suggest the combined evaluation of liquid biopsy and radiomic features for obtaining minimally invasive and cost-effective biomarkers in the first-line clinical setting of NSCLC undergoing first-line pembrolizumab.

Lung cancer represents the leading cause of cancer-related mortality worldwide, with around 1.8 million deaths in 2020. For this reason, there is an enormous interest in finding early diagnostic tools and novel therapeutic approaches, one of which is extracellular vesicles (EVs). EVs are nanoscale membranous particles that can carry proteins, lipids, and nucleic acids (DNA and RNA), mediating various biological processes, especially in cell–cell communication. As such, they represent an interesting biomarker for diagnostic analysis that can be performed easily by liquid biopsy. Moreover, their growing dataset shows promising results as drug delivery cargo. The aim of our work is to summarize the recent advances in and possible implications of EVs for early diagnosis and innovative therapies for lung cancer.

An amine derivative of hyaluronic acid (HA) was crosslinked to obtain a 3D dried sponge. The sponge was subsequently rehydrated using secretome from human mesenchymal stromal cells (MSCs), resulting in the formation of a hydrogel. The release kinetics analysis demonstrated that the hydrogel effectively sustained secretome release, with 70% of the initially loaded wound-healing-associated cytokines being released over a 12-day period. Tuning the hydrogel properties through heparin crosslinking resulted in a biomaterial with a distinct mechanism of action. Specifically, the presence of heparin enhanced water uptake capacity of the hydrogel and increased its sensitivity to enzymatic degradation. Notably, the heparin crosslinking also led to a significant retention of cytokines within the hydrogel matrix. Overall, the secretome-rehydrated HA hydrogel holds promise as a versatile device for regenerative medicine applications: the non-heparinized hydrogel may function as a biomaterial with low reabsorption rates, sustaining the release of bioactive molecules contained in MSC secretome. In contrast, the heparinized hydrogel may serve as a depot of bioactive molecules with faster reabsorption rates. Given its patch-like characteristic, the HA-based hydrogel appears suitable as topical treatment for external organs, such as the skin.

e14553 Background: In the liquid biopsy era, the interrogation of extracellular vesicles (EVs) content might be complementary for cancer prognostication and response assessment. In this explorative study, we aimed to describe whether the serial characterization of BCEVs plasma tumor-derived EVs could longitudinally reflect response and resistance to available first-line treatments, investigating the potential to predict clinical outcomes in patients with advanced NSCLC (aNSCLC). Methods: From 2020/02 to 2022/05, 27 treatment-naïve patients with aNSCLC (stageIIIB/IV), eligible for first-line treatment with an EGFRTKI (Osimertinib) or ICIs (e.g. Pembrolizumab ± platinum‐based chemotherapy) based on the predictive molecular pathology, were enrolled. Blood samples (∼5 mL) were collected into K2EDTA tubes early in the morning with fasting condition. We isolated EVs from 2 ml of plasma using exo-Easy Maxi Kit (Qiagen-USA). EVs’s size and distribution was determine by use of Dynamic Light Scattering method (DLS). Besides a Bradford assay (cfEV) for the indirect quantification of EV amount based on EVs proteins content was performed. According to the radiologic response, EV analyses were evaluated in patients with available plasma samples from baseline (T0) to the first radiologic evaluation of disease within 12 weeks (T1). Results: From 2020/02 to 2022/05, 27treatment-naïve patients with aNSCLC (stageIIIB/IV), eligible for first-line treatment with an EGFRTKI (Osimertinib) or ICIs (e.g. Pembrolizumab ± platinum‐based chemotherapy) based on the predictive molecular pathology, were enrolled. Blood samples (∼5 mL) were collected into K2EDTA tubes early in the morning with fasting condition. We isolated EVs from 2 ml of plasma using exo-Easy Maxi Kit (Qiagen-USA). EVs’s size and distribution was determine by use of Dynamic Light Scattering method (DLS). Besides a Bradford assay (cfEV) for the indirect quantification of EV amount based on EVs proteins content was performed. According to the radiologic response, EV analyses were evaluated in patients with available plasma samples from baseline (T0) to the first radiologic evaluation of disease within 12 weeks (T1). Conclusions: This real-world study confirmed the in vivo feasibility of isolating and characterizing BCEVs via DLS and Bradford assay from the plasma of patients with ANSCLC. The quantitative changes of the plasma EVs correlated with survival in the real-time monitoring of such first-line patients, mostly in the oncogene-addicted population receiving osimertinib.

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

10592 Background: Testing for BRCA mutations (BRCAm) and genomic instability can identify epithelial ovarian cancer (OC) patients most likely to benefit from PARP-inhibitors (PARPi). However, current biomarkers of non- BRCA Homologous Recombination Repair (HRR) mutations are insufficient for guiding use of PARPi in the clinic. Despite non- BRCA HRR pathway gene mutations are rare, these patients may benefit from PARPi. Furthermore, recent preclinical findings showed that sensitivity to PARPi could be associated also with mutations in mismatch repair (MMR) genes, although sensitivity in the clinic is not proven. Methods: We report our real-life experience to assess the HRD status beyond BRCAm in newly-diagnosed OC patients, with the use of HRR gene panel and HRD genomic instability tests. After primary diagnosis, tumor and germline BRCA (g BRCA) status were assessed and, if BRCA WT, tumor HRR deficiency status were centrally determined by myChoiceCDx (Myriad) assay. NGS panel evaluating 20 MMR and HRR-genes beyond BRCA1/2 was proposed to patients with significant personal and/or family history of cancer, resulted negative to g BRCA testing. Results: From January 2017 to January 2023, 540 unselected epithelial OC patients, aging 27 to 81, were tested for tumor and g BRCA status; 109 were carriers of germline pathogenic or likely pathogenic variants (PVs) in BRCA1/2 genes (20.2%): 72 in BRCA1 (66.1%) and 37 in BRCA2 gene (33.9%). Additional 19 patients showed somatic PVs confined to tumor samples (3.5%).In the population of 70 BRCA WT patients tested by multigene panel testing, 14 germline PVs in HRR-associated (7.1%) or MMR-associated genes (12.8%) were found, including 5 in MUTYH (35.7%), 2 in ATM (14.3%), 2 in MLH1 (14.3%), 2 in PMS2 (14.3%), 1 in RAD51C (7.14%), 1 in RAD51D (7.14%), and 1 in CHEK2 gene (7.14%).Out of 72 samples analyzed by HRD genomic instability test, 23 cases were identified as HRD positive (31.9%), with a median GIS score of 65.5 (44-83). Median GIS were 35 (1-72) in the 6 cases of non- BRCA mutated tumors (8.3%), 40 (4-82) in the 17 tumor with non- BRCA variant of uncertain significance (VUS) (23.6%), and 27 (2-83) in the WT samples. Median GIS were significantly higher in tumors with PVs/VUS of RAD51 than BRIP1 (55.5 vs 35). Conclusions: HRRm gene panel and HRD genomic instability tests are not interchangeable to study HRR deficiency beyond BRCAm. HRD genomic instability test is effective to identify patients most likely to gain benefit from PARPi, but not for predicting familial risk of cancer. In our population, NGS panel evaluating HRR genes, beyond BRCA1/2, improved the detection rate of HRRm by 7%, with additional finding of MMR germline mutations, and important clinical implications for family members. Prospective data are expected to evaluate the effectiveness of PARPi in these population.

10519 Background: BRCA1/2 genes ( BRCA) play a prominent role in the Homologous Recombination Repair (HRR) pathway. Following the technological progress and deeper knowledge on BRCA-related cancers, the demand for genetic testing is rapidly increasing. Beyond BRCA1/2, other genes are involved in the HRR, including ATM, PALB2, RAD51, and BARD1. Due to the important role in the cellular repair process, deleterious variants in HRR genes may cause inadequate DNA damage repair in cardiomyocytes. The role of BRCA1/2 as predisposing condition to cardiac dysfunction is debated, and the contribution by no- BRCA genes is still unknown. Methods: This is a multicenter, retrospective, study to investigate the risk of heart-insults from anthracyclines on adjuvant setting in BC patients carrying germline pathogenic or likely pathogenic variant (PV) (classes IV and V) in BRCA and no- BRCA HRR pathway genes. We collected genetic and clinical data, and evaluated the left ventricular ejection fraction (LVEF) at cardiac ultrasound, before starting ACR therapy, and at subsequent time points, according to clinical indications. Results: Three hundred and sixty (360) BC patients, aging 22 to 80, were included in this study; 131 patients were carriers of germline PVs in HRR pathway genes: 52 in BRCA1 gene (39.7%), 48 in BRCA2 gene (36.6%), and 31 harbored PVs in no- BRCA HRR pathway genes (23.7%), including PVs in PALB2, CHEK2, ATM, RAD51C, RAD50 and BARD1 genes. In the cohort of 229 patients without PVs, 47 showed variant of uncertain significance (VUS, class III), and 173 had genetic testing not informative. When LVEF between the groups was compared, the difference was not significant for the pre-treatment values. Notably, individuals carrying BRCA or other HRR gene deleterious variants, showed a statistically significant reduction of LVEF > 5% at the second time-point (3 month), compared to the LVEF pre-treatment values (p = 0.001). A marked LVEF reduction was in mutated patients treated with risk-reducing bilateral salpingo-oophorectomy prior to age 40, body mass index > 25, and type-II diabetes mellitus. The latter risk factor was probably related to increased risk developing insulin-resistance reported for BRCA-mutated patients. Conclusions: Our data suggest that PVs in BRCA or other genes involved in HRR pathway, can lead to impaired homologous recombination, thus increasing sensitivity of cardiac cells to DNA damaging chemotherapy in BC patients. In this subgroup of patients, other measurements such as the global longitudinal strain (GLS), and a more in-depth assessment of risk factors, could be proposed to optimize cardiovascular risk-management and to improve long-term survival.

Bone is a highly vascularized tissue and relies on the angiogenesis and response of cells in the immediate environmental niche at the defect site for regeneration. Hence, the ability to control angiogenesis and cellular responses during osteogenesis has important implications in tissue-engineered strategies. Self-assembling ionic-complementary peptides have received much interest as they mimic the natural extracellular matrix. Three-dimensional (3D)-printed biphasic calcium phosphate (BCP) scaffolds coated with self-assembling DAR 16-II peptide provide a support template with the ability to recruit and enhance the adhesion of cells. In vitro studies demonstrated prompt the adhesion of both human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC), favoring endothelial cell activation toward an angiogenic phenotype. The SEM-EDS and protein micro bicinchoninic acid (BCA) assays demonstrated the efficacy of the coating. Whole proteomic analysis of DAR 16-II-treated HUVECs demonstrated the upregulation of proteins involved in cell adhesion (HABP2), migration (AMOTL1), cytoskeletal re-arrangement (SHC1, TMOD2), immuno-modulation (AMBP, MIF), and morphogenesis (COL4A1). In vivo studies using DAR-16-II-coated scaffolds provided an architectural template, promoting cell colonization, osteogenesis, and angiogenesis. In conclusion, DAR 16-II acts as a proactive angiogenic factor when adsorbed onto BCP scaffolds and provides a simple and effective functionalization step to facilitate the translation of tailored 3D-printed BCP scaffolds for clinical applications.

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.