Background Increased fetal nuchal translucency (NT ≥ 3.5mm) is a well-established ultrasound marker associated with an increased risk of genomic alterations. CGH array analysis has enhanced the detection of copy number variations (CNVs), but also of variants of uncertain significance (VUS), complicating interpretation and genetic counselling. Methods We retrospectively analyzed prenatal data from two Italian laboratories on 1,104 fetuses with isolated increased NT to assess the frequency, distribution, and clinical interpretation of pathogenic CNVs and VUS across two laboratories over a 15-year period. Results A total of 118 genomic abnormalities were identified. CGH array detected 37 pathogenic CNVs and 71 VUS. Pathogenic CNVs showed a non-random distribution, with enrichment on chromosomes 22, 1, 2, and X, while VUS were more widely dispersed across the genome. In ten cases, CGH array findings led to additional conventional cytogenetic investigations, revealing structural rearrangements, including complex and unbalanced translocations and multiple aneuploidies. In fetuses with isolated increased NT, CGH array adds approximately 3.4% (37/1,104) detection of pathogenic CNVs beyond karyotyping, but also generates a 6.4% (71/1,104) of VUS. Subsequent reclassification of variants revealed that about 62% of CNVs previously classified as pathogenic are now considered VUS or benign, highlighting the evolving nature of genomic interpretation. Regarding CNVs classified as VUSs, the 3% are now classified as likely pathogenic, while 64% are likely benign. Conclusion Integration of molecular and conventional cytogenetic approaches improves diagnostic accuracy and is essential for comprehensive prenatal genetic counselling, but CNV findings need to be continuously evaluated for possible evolution in their interpretation.

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults, with its significant inter- and intra-tumoral heterogeneity being a major factor in its treatment resistance and overall prognosis. GB diagnosis typically involves magnetic resonance imaging, confirmed by histology after surgical resection or biopsy. Recurrence is almost expected despite adjuvant therapies. Extracellular vesicles (EVs) may represent promising cancer biomarkers for diagnosis, prognosis, and therapeutic monitoring. In this work, we monitored 21 GB patients at different time intervals performing a quantitative and dimensional analysis of plasma-derived EVs, with the aim of finding correlations with their clinical course. Our analyses revealed a slight correlation with patients’ clinical conditions during follow-up, such as tumor time recurrence over time, but no significant difference in plasma EV concentration in GB patients and healthy control subjects (HC), contrary to previously published data. Although based on a limited number of patients, our methodological study highlights the need for a universal analysis method to compare data from large patient populations in order to use EVs as a biomarker for the diagnosis of recurrence by liquid biopsy, especially in GB, a tumor known for its heterogeneity.

Chromosomal triplications are rare structural variations often associated with complex phenotypes. We report the molecular characterization of a novel intrachromosomal triplication at 18q12.1q21.2 identified in a fetus with ultrasound abnormalities. Conventional karyotyping and array-CGH revealed a partial tetrasomy and a 26 Mb region of loss of homozygosity (LOH), extending from the triplication to the telomere. Long-read sequencing (LRS) identified breakpoint junctions revealing a direct-inverted-direct triplication structure. Breakpoint analysis suggested that this rearrangement arose through a U-type exchange between sister chromatids, likely mediated by microhomology-based mechanisms. This process likely generated a transient dicentric chromosome that subsequently broke during mitosis. The resulting duplicated chromosome may have been stabilized by telomere capture, consistent with the triplicated 18q12.1q21.2 region followed by the 18q21.2q23 LOH. Nine genes within the triplicated region, including SMAD2 and SMAD4, showed high predicted sensitivity to increased dosage, possibly contributing to the clinical phenotype. This study highlights the utility of LRS in defining complex chromosomal rearrangements and emphasizes the importance of molecular breakpoint analysis for understanding pathogenic mechanisms and improving genetic prenatal diagnosis.

This study reports the design, synthesis, and biological evaluation of novel inhibitors targeting the epigenetic enzymes ALKBH2 and ALKBH5 as potential adjuvants to temozolomide therapy in glioblastoma. Given their critical role in DNA/RNA demethylation, tumor progression, and drug resistance, their inhibition represents a promising therapeutic strategy. Building on the previously identified lead compound MV1035, we employed structure-based drug design to develop new derivatives, including a second-generation series incorporating a fumarate hydrazide moiety to enhance binding affinity through interaction with both substrate- and cofactor-binding sites. Molecular docking studies predicted significantly improved binding for a set of new compounds but, due to multiple synthetic drawbacks, only a subset of the designed series was synthesized and evaluated biologically. MV3030 emerged as the most promising candidate. MV3030 demonstrated an inhibitory effect on ALKBH2 comparable to MV1035, also showing a more moderate inhibitory effect on ALKBH5. Notably, it exhibited intrinsic cytotoxicity in U87-MG cells and patient-derived glioma stem cells, whereas normal astrocytes exhibited markedly higher resistance to the treatment. Furthermore, MV3030 enhanced temozolomide efficacy and displayed favorable blood-brain barrier permeability both in silico and in vitro. Moreover, MV3030 modulated the FoxM1/Wnt/β-catenin axis. Overall, these findings identify MV3030 as a promising compound with the potential to overcome temozolomide resistance and improve glioblastoma treatment.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a short survival time after standard therapy administration including radiotherapy (RT) associated with temozolomide (TMZ). Here, we investigated the effects of radiochemotherapy in association with metformin (MET), a drug targeting cell metabolism on a syngeneic GBM mouse model using Positron Emission Tomography imaging with [18F]FLT and [18F]VC701 and single-cell RNA-sequencing analysis. The addition of drugs to RT significantly increased survival and [18F]FLT showed an early predictive response of combined therapy. We identified the presence of heterogeneous tumor populations with different treatment sensitivity and a complex immune evasive microenvironment. Tumor cells surviving to treatments showed immune response, among the main differentially modulated biological functions and a potential role of long non-coding RNAs (lncRNAs) in treatment resistance. Association with TMZ or TMZ plus MET reduced the pro-tumor phenotype of immune reaction acting more on myeloid cells the first and on lymphocytes the latter. Off note, MET add-on counteracted the immune-evasive phenotype particularly of T cells suggesting a potential role of MET also in adopted immunity.

The blood-brain barrier (BBB) is a highly selective barrier that strictly controls the passage of substances and cells into the brain, protecting it from potential harm while preserving homeostasis. It is composed of specialised endothelial cells (ECs), along with surrounding cells, such as pericytes and astrocytes. In glioblastoma (GBM), the most prevalent primary malignant brain tumour in adults, the BBB is heterogeneously dysfunctional. In the tumour microenvironment, regions enriched with glioblastoma stem cells (GSCs) are protected by an intact BBB. However, the influence of GSCs on BBB function remains largely unexplored. In this study, the impact of patient-derived GSC (PD GSC) secretomes on human brain capillary ECs has been investigated in vitro. Results showed that secretomes decrease the BBB permeability, leading to an increase of transendothelial electrical resistance and of tight junction protein claudin-5 (CLDN5) levels. Moreover, the receptor for advanced glycation endproducts (RAGE), which is involved in cancer and chemotherapy resistance, modulates CLDN5 expression by activating the pERK/ERK signaling pathway and influences junctional organization. These findings suggest a functional pathway through which PD GSC secretomes can modulate BBB permeability, potentially impacting therapeutic efficacy.

The vacuolar proton-translocating ATPase (V-ATPase) is a transmembrane multi-protein complex fundamental in maintaining a normal intracellular pH. In the tumoral contest, its role is crucial since the metabolism underlying carcinogenesis is mainly based on anaerobic glycolytic reactions. Moreover, neoplastic cells use the V-ATPase to extrude chemotherapy drugs into the extra-cellular compartment as a drug resistance mechanism. In glioblastoma (GBM), the most malignant and incurable primary brain tumor, the expression of this pump is upregulated, making it a new possible therapeutic target. In this work, the bafilomycin A1-induced inhibition of V-ATPase in patient-derived glioma stem cell (GSC) lines was evaluated together with temozolomide, the first-line therapy against GBM. In contrast with previous published data, the proposed treatment did not overcome resistance to the standard therapy. In addition, our data showed that nanomolar dosages of bafilomycin A1 led to the blockage of the autophagy process and cellular necrosis, making the drug unusable in models which are more complex. Nevertheless, the increased expression of V-ATPase following bafilomycin A1 suggests a critical role of the proton pump in GBM stem components, encouraging the search for novel strategies to limit its activity in order to circumvent resistance to conventional therapy.

Background/Objectives: Aneuploidy is a prevalent cancer feature that occurs in many solid tumors. For example, high-grade serous ovarian cancer shows a high level of copy number alterations and genomic rearrangements. This makes genomic variants appealing as diagnostic or prognostic biomarkers, as well as for their easy detection. In this study, we focused on copy number (CN) losses shared by ovarian cancer stem cells (CSCs) to identify chromosomal regions that may be important for CSC features and, in turn, for patients’ prognosis. Methods: Array-CGH and bioinformatic analyses on three CSCs subpopulations were performed. Results: Pathway and gene ontology analyses on genes involved in copy number loss in all CSCs revealed a significant decrease in mRNA surveillance pathway, as well as miRNA-mediated gene silencing. Then, starting from these CN losses, we validated their potential prognostic relevance by analyzing the TCGA cohort. Notably, losses of 4q34.3-q35.2, 8p21.2-p21.1, and 18q12.2-q23 were linked to increased genomic instability. Loss of 18q12.2-q23 was also related to a higher tumor stage and poor prognosis. Finally, specific genes mapping in these regions, such as PPP2R2A and TPGS2A, emerged as potential biomarkers. Conclusions: Our findings highlight the importance of genomic alterations in ovarian cancer and their impact on tumor progression and patients’ prognosis, offering advance in understanding of the application of numerical aberrations as prognostic ovarian cancer biomarkers.

When an increased nuchal translucency (>3.00 mm) is observed during the echographic examination of a foetus in the first trimester of pregnancy, an increased risk of chromosomopathy is considered, and the pregnant woman is offered the possibility of an invasive investigation. Here, we focused our attention on prenatal diagnosis issues in cases of foetuses with cytogenetically balanced reciprocal translocations. We report the finding of a cytogenetically balanced, de facto genomically unbalanced translocation that poses a challenge in a case of prenatal diagnosis, changing the risk of Down syndrome in a Zellweger syndromic spectrum risk (PEX3 deletion). At term, a healthy baby was born. This case teaches that prenatal diagnosis in cases of foetuses at increased risk of chromosomal abnormality imperatively requires molecular investigation in addition to a morphological karyotype.

Glioblastoma (GBM) is the most aggressive and invasive primary brain tumor. Current therapies are not curative, and patients' outcomes remain poor with an overall survival of 20.9 months after surgery. The typical growing pattern of GBM develops by infiltrating the surrounding apparent normal brain tissue within which the recurrence is expected to appear in the majority of cases. Thus, in the last decades, an increased interest has developed to investigate the cellular and molecular interactions between GBM and the peritumoral brain zone (PBZ) bordering the tumor tissue. The aim of this review is to provide up-to-date knowledge about the oncogenic properties of the PBZ to highlight possible druggable targets for more effective treatment of GBM by limiting the formation of recurrence, which is almost inevitable in the majority of patients. Starting from the description of the cellular components, passing through the illustration of the molecular profiles, we finally focused on more clinical aspects, represented by imaging and radiological details. The complete picture that emerges from this review could provide new input for future investigations aimed at identifying new effective strategies to eradicate this still incurable tumor.

Despite the efforts made in recent decades, glioblastoma is still the deadliest primary brain cancer without cure. The potential role in tumour maintenance and progression of the peritumoural brain zone (PBZ), the apparently normal area surrounding the tumour, has emerged. Little is known about this area due to a lack of common definition and due to difficult sampling related to the functional role of peritumoural healthy brain. The aim of this work was to better characterize the PBZ and to identify genes that may have role in its malignant transformation. Starting from our previous study on the comparison of the genomic profiles of matched tumour core and PBZ biopsies, we selected CDK4 and EXT2 as putative malignant drivers of PBZ. The gene expression analysis confirmed their over-expression in PBZ, similarly to what happens in low-grade glioma and glioblastoma, and CDK4 high levels seem to negatively influence patient overall survival. The prognostic role of CDK4 and EXT2 was further confirmed by analysing the TCGA cohort and bioinformatics prediction on their gene networks and protein–protein interactions. These preliminary data constitute a good premise for future investigations on the possible role of CDK4 and EXT2 in the malignant transformation of PBZ.

Complex chromosomal rearrangements are rare events compatible with survival, consisting of an imbalance and/or position effect of one or more genes, that contribute to a range of clinical presentations. The investigation and diagnosis of these cases are often difficult. The interpretation of the pattern of pairing and segregation of these chromosomes during meiosis is important for the assessment of the risk and the type of imbalance in the offspring. Here, we investigated two unrelated pediatric carriers of complex rearrangements of chromosome 7. The first case was a 2-year-old girl with a severe phenotype. Conventional cytogenetics evidenced a duplication of part of the short arm of chromosome 7. By array-CGH analysis, we found a complex rearrangement with three discontinuous trisomy regions (7p22.1p21.3, 7p21.3, and 7p21.3p15.3). The second case was a newborn investigated for hypodevelopment and dimorphisms. The karyotype analysis promptly revealed a structurally altered chromosome 7. The array-CGH analysis identified an even more complex rearrangement consisting of a trisomic region at 7q11.23q22 and a tetrasomic region of 4.5 Mb spanning 7q21.3 to q22.1. The mother's karyotype examination revealed a complex rearrangement of chromosome 7: the 7q11.23q22 region was inserted in the short arm at 7p15.3. Finally, array-CGH analysis showed a trisomic region that corresponds to the tetrasomic region of the son. Our work proved that the integration of several technical solutions is often required to appropriately analyze complex chromosomal rearrangements in order to understand their implications and offer appropriate genetic counseling.

The largest multi-gene family in metazoans is the family of olfactory receptor (OR) genes. Human ORs are organized in clusters over most chromosomes and seem to include >0.1% the human genome. Because 369 out of 856 OR genes are mapped on chromosome 11 (HSA11), we sought to determine whether they mediate structural rearrangements involving this chromosome. To this aim, we analyzed 220 specimens collected during diagnostic procedures involving structural rearrangements of chromosome 11. A total of 222 chromosomal abnormalities were included, consisting of inversions, deletions, translocations, duplications, and one insertion, detected by conventional chromosome analysis and/or fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (arrayCGH). We verified by bioinformatics and statistical approaches the occurrence of breakpoints in cytobands with or without OR genes. We found that OR genes are not involved in chromosome 11 reciprocal translocations, suggesting that different DNA motifs and mechanisms based on homology or non-homology recombination can cause chromosome 11 structural alterations. We also considered the proximity between the chromosomal territories of chromosome 11 and its partner chromosomes involved in the translocations by using the deposited Hi-C data concerning the possible occurrence of chromosome interactions. Interestingly, most of the breakpoints are located in regions highly involved in chromosome interactions. Further studies should be carried out to confirm the potential role of chromosome territories' proximity in promoting genome structural variation, so fundamental in our understanding of the molecular basis of medical genetics and evolutionary genetics.

The lack of effective screening and successful treatment contributes to high ovarian cancer mortality, making it the second most common cause of gynecologic cancer death. Development of chemoresistance in up to 75% of patients is the cause of a poor treatment response and reduced survival. Therefore, identifying potential and effective biomarkers for its diagnosis and prognosis is a strong critical need. Copy number alterations are frequent in cancer, and relevant for molecular tumor stratification and patients' prognoses. In this study, array-CGH analysis was performed in three cell lines and derived cancer stem cells (CSCs) to identify genes potentially predictive for ovarian cancer patients' prognoses. Bioinformatic analyses of genes involved in copy number gains revealed that AhRR and PPP1R3C expression negatively correlated with ovarian cancer patients' overall and progression-free survival. These results, together with a significant association between AhRR and PPP1R3C expression and ovarian cancer stemness markers, suggested their potential role in CSCs. Furthermore, AhRR and PPP1R3C's increased expression was maintained in some CSC subpopulations, reinforcing their potential role in ovarian cancer. In conclusion, we reported for the first time, to the best of our knowledge, a prognostic role of AhRR and PPP1R3C expression in serous ovarian cancer.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a malignant prognosis. GBM is characterized by high cellular heterogeneity and its progression relies on the interaction with the central nervous system, which ensures the immune-escape and tumor promotion. This interplay induces metabolic, (epi)-genetic and molecular rewiring in both domains. In the present study, we aim to characterize the time-related changes in the GBM landscape, using a syngeneic mouse model of primary GBM. GL261 glioma cells were injected in the right striatum of immuno-competent C57Bl/6 mice and animals were sacrificed after 7, 14, and 21 days (7D, 14D, 21D). The tumor development was assessed through 3D tomographic imaging and brains were processed for immunohistochemistry, immunofluorescence, and western blotting. A human transcriptomic database was inquired to support the translational value of the experimental data. Our results showed the dynamic of the tumor progression, being established as a bulk at 14D and surrounded by a dense scar of reactive astrocytes. The GBM growth was paralleled by the impairment in the microglial/macrophagic recruitment and antigen-presenting functions, while the invasive phase was characterized by changes in the extracellular matrix, as shown by the analysis of tenascin C and metalloproteinase-9. The present study emphasizes the role of the molecular changes in the microenvironment during the GBM progression, fostering the development of novel multi-targeted, time-dependent therapies in an experimental model similar to the human disease.

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Improvements in microarray-based comparative genomic hybridization technology have allowed for high-resolution detection of genome wide copy number alterations, leading to a better definition of rearrangements and supporting the study of pathogenesis mechanisms. In this study, we focused our attention on chromosome 8p. We report 12 cases of 8p rearrangements, analyzed by molecular karyotype, evidencing a continuum of fragility that involves the entire short arm. The breakpoints seem more concentrated in three intervals: one at the telomeric end, the others at 8p23.1, close to the beta-defensin gene cluster and olfactory receptor low-copy repeats. Hypothetical mechanisms for all cases are described. Our data extend the cohort of published patients with 8p aberrations and highlight the need to pay special attention to these sequences due to the risk of formation of new chromosomal aberrations with pathological effects.

Purpose Bladder cancer is the most common malignancy of the urinary tract and one of the most prevalent cancers worldwide. It represents a spectrum of diseases, from recurrent non-invasive tumors (NMIBCs) managed chronically, to muscle infiltrating and advanced-stage disease (MIBC) that requires multimodal and invasive treatment. Multiple studies have underlined the complexity of bladder tumors genome, highlighting many specific genetic lesions and genome-wide occurrences of copy-number alterations (CNAs). In this study, we analyzed CNAs of selected genes in our cohorts of cancer stem cells (CSCs) and in The Cancer Genome Atlas (TCGA-BLCA) cohort with the aim to correlate their frequency with patients’ prognosis. Methods CNAs have been verified on our array-CGH data previously reported on 19 bladder cancer biopsies (10 NMIBCs and 9 MIBCs) and 16 matched isolated CSC cultures. In addition, CNAs data have been consulted on the TCGA database, to search correlations with patients’ follow-up. Finally, mRNA expression levels of LRP1B in TGCA cohort were obtained from The Human Protein Atlas. Results We firstly identified CNAs differentially represented between TGCA data and CSCs derived from NMIBCs and MIBCs, and we correlated the presence of these CNAs with patients’ follow-up. LRP1B loss was significantly increased in CSCs and linked to short-term poor prognosis, both at genomic and transcriptomic level, confirming its pivotal role in bladder cancer tumorigenesis. Conclusion Our study allowed us to identify potential "predictive" prognostic CNAs for bladder cancer, implementing knowledge for the ultimate goal of personalized medicine.

The study of glioblastoma genomic profiles permits the use of CNAs load to identify patients with poor prognosis and the understanding of the genomic signatures essential for the disease maintenance and the identification of new potential biomarkers.

Recent evidences suggest the involvement of the Vacuolar H+ ATPase (V-ATPase) in the development and/or progression of Glioblastoma Multiforme (GBM). This proton pump could be a valid therapeutic target but more in-depth studies are necessary. The aim of this study is to better define the in vitro effects on Glioma Stem Cell (GSC) primary cultures viability of single and combined treatment with Bafilomycin-A1 (Baf-A1), a V-ATPase inhibitor, and Temozolomide (TMZ), the chemotherapeutic agent currently used to treat GBM patients. We found out that GSC were resistant to TMZ and more sensitive to treatments with Baf-A1 and that the two drugs exerted a synergistic effect when administered together.