Vacuolar Proton-Translocating ATPase May Take Part in the Drug Resistance Phenotype of Glioma Stem Cells.

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.

Differential Expression of NOTCH-1 and Its Molecular Targets in Response to Metronomic Followed by Conventional Therapy in a Patient with Advanced Triple-Negative Breast Cancer.

A group of 27 patients diagnosed with metastatic triple-negative breast cancer (mTNBC) was randomly distributed into two groups and underwent different lines of metronomic treatment (mCHT). The former group (N 14) received first-line mCHT and showed a higher overall survival rate than the second group (N 13), which underwent second-line mCHT. Analysis of one patient still alive from the first group, diagnosed with mTNBC in 2019, showed a complete metabolic response (CMR) after a composite approach implicating first-line mCHT followed by second-line epirubicin and third-line nab-paclitaxel, and was chosen for subsequent molecular characterization. We found altered expression in the cancer stemness-associated gene NOTCH-1 and its corresponding protein. Additionally, we found changes in the expression of oncogenes, such as MYC and AKT, along with their respective proteins. Overall, our data suggest that a first-line treatment with mCHT followed by MTD might be effective by negatively regulating stemness traits usually associated with the emergence of drug resistance.

High-sensitivity analysis of clonal hematopoiesis reveals increased clonal complexity of potential-driver mutations in severe COVID-19 patients.

Whether Clonal Hematopoiesis (CH) represents a risk factor for severity of the COVID-19 disease remains a controversial issue. We report the first high- sensitivity analysis of CH in COVID-19 patients (threshold of detection at 0.5% vs 1 or 2% in previous studies). We analyzed 24 patients admitted to ICU for COVID-19 (COV-ICU) and 19 controls, including healthy subjects and asymptomatic SARS-CoV2-positive individuals. Despite the significantly higher numbers of CH mutations identified (80% mutations with <2% variant allele frequency, VAF), we did not find significant differences between COV-ICU patients and controls in the prevalence of CH or in the numbers, VAF or functional categories of the mutated genes, suggesting that CH is not overrepresented in patients with COVID-19. However, when considering potential drivers CH mutations (CH-PD), COV-ICU patients showed higher clonal complexity, in terms of both mutation numbers and VAF, and enrichment of variants reported in myeloid neoplasms. However, we did not score an impact of increased CH-PD on patient survival or clinical parameters associated with inflammation. These data suggest that COVID-19 influence the clonal composition of the peripheral blood and call for further investigations addressing the potential long-term clinical impact of CH on people experiencing severe COVID-19. We acknowledge that it will indispensable to perform further studies on larger patient cohorts in order to validate and generalize our conclusions. Moreover, we performed CH analysis at a single time point. It will be necessary to consider longitudinal approaches with long periods of follow-up in order to assess if the COVID-19 disease could have an impact on the evolution of CH and long-term consequences in patients that experienced severe COVID-19.

Olfactory receptor genes and chromosome 11 structural aberrations: Players or spectators?

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 (array-CGH). 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.

TWIST1 Upregulation Is a Potential Target for Reversing Resistance to the CDK4/6 Inhibitor in Metastatic Luminal Breast Cancer Cells.

Cyclin-dependent kinase (CDK) 4/6 inhibitors have significantly improved progression-free survival in hormone-receptor-positive (HR+), human-epidermal-growth-factor-receptor-type-2-negative (HER2-) metastatic luminal breast cancer (mLBC). Several studies have shown that in patients with endocrine-sensitive or endocrine-resistant LBC, the addition of CDK4/6 inhibitors to endocrine therapy significantly prolongs progression-free survival. However, the percentage of patients who are unresponsive or refractory to these therapies is as high as 40%, and no reliable and reproducible biomarkers have been validated to select a priori responders or refractory patients. The selection of mutant clones in the target oncoprotein is the main cause of resistance. Other mechanisms such as oncogene amplification/overexpression or mutations in other pathways have been described in several models. In this study, we focused on palbociclib, a selective CDK4/6 inhibitor. We generated a human MCF-7 luminal breast cancer cell line that was able to survive and proliferate at different concentrations of palbociclib and also showed cross-resistance to abemaciclib. The resistant cell line was characterized via RNA sequencing and was found to strongly activate the epithelial-to-mesenchymal transition. Among the top deregulated genes, we found a dramatic downregulation of the CDK4 inhibitor CDKN2B and an upregulation of the TWIST1 transcription factor. TWIST1 was further validated as a target for the reversal of palbociclib resistance. This study provides new relevant information about the mechanisms of resistance to CDK4/6 inhibitors and suggests potential new markers for patients' follow-up care during treatment.

Progression after First-Line Cyclin-Dependent Kinase 4/6 Inhibitor Treatment: Analysis of Molecular Mechanisms and Clinical Data.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6iss) are widely used in first-line metastatic breast cancer. For patients with progression under CDK4/6is, there is currently no standard treatment recommended at the category 1 level in international guidelines. The purpose of this article is to review the cellular mechanisms underlying the resistance to CDK4/6is, as well as treatment strategies and the clinical data about the efficacy of subsequent treatments after CDK4/6is-based therapy. In the first part, this review mainly discusses cell-cycle-specific and cell-cycle-non-specific resistance to CDK4/6is, with a focus on early and late progression. In the second part, this review analyzes potential therapeutic approaches and the available clinical data on them: switching to other CDK4/6is, to another single hormonal therapy, to other target therapies (PI3K, mTOR and AKT) and to chemotherapy.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

Evaluating [18F]FDG and [18F]FLT Radiotracers as Biomarkers of Response for Combined Therapy Outcome in Triple-Negative and Estrogen-Receptor-Positive Breast Cancer Models.

Breast cancer (BC) is the most frequent cancer and the second leading cause of death in women. A typical feature of BC cells is the metabolic shift toward increased glycolysis, which has become an interesting therapeutic target for metabolic drugs such as metformin (MET). Recently, the administration of the antihypertensive syrosingopine (SYRO) in combination with MET has shown a synergistic effect toward a variety of cancers. However, a fundamental need remains, which is the development of in vivo biomarkers that are able to detect early clinical response. In this study, we exploited a triple-negative murine BC cell line (4T1) and a metastatic ER+ murine BC cell line (TS/A) in order to investigate, in vivo, the early response to treatment, based on MET and/or SYRO administration, evaluating [18F]FDG and [18F]FLT as potential biomarkers via PET/CT. The study provides evidence that SYRO plus MET has a synergistic effect on tumor growth inhibition in both 4T1 and TS/A experimental models and has showed the highest efficacy on the TNBC xenograft mice (4T1) via the expression reduction in the lactate transporter MCT4 and in the epithelial-mesenchymal transition biomarker Snail, promoting its potential application in therapy settings. In addition, the selective reduction in the [18F]FLT tumor uptake (at 7 dd), observed in the SYRO plus MET treated mice in comparison with the vehicle group, suggests that this radiotracer could be potentially used as a biomarker for the early detection of therapy response, in both evaluated xenografts models.

Influence of Bruton's Tyrosine Kinase (BTK) on Epithelial-Mesenchymal Transition (EMT) Processes and Cancer Stem Cell (CSC) Enrichment in Head and Neck Squamous Cell Carcinoma (HNSCC).

Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.

Genomic Complexity and Complex Chromosomal Rearrangements in Genetic Diagnosis: Two Illustrative Cases on Chromosome 7.

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.

AhRR and PPP1R3C: Potential Prognostic Biomarkers for Serous Ovarian Cancer.

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.

Pathologist Validation of a Machine Learning-Derived Feature for Colon Cancer Risk Stratification.

Importance: Identifying new prognostic features in colon cancer has the potential to refine histopathologic review and inform patient care. Although prognostic artificial intelligence systems have recently demonstrated significant risk stratification for several cancer types, studies have not yet shown that the machine learning-derived features associated with these prognostic artificial intelligence systems are both interpretable and usable by pathologists. Objective: To evaluate whether pathologist scoring of a histopathologic feature previously identified by machine learning is associated with survival among patients with colon cancer. Design, Setting, and Participants: This prognostic study used deidentified, archived colorectal cancer cases from January 2013 to December 2015 from the University of Milano-Bicocca. All available histologic slides from 258 consecutive colon adenocarcinoma cases were reviewed from December 2021 to February 2022 by 2 pathologists, who conducted semiquantitative scoring for tumor adipose feature (TAF), which was previously identified via a prognostic deep learning model developed with an independent colorectal cancer cohort. Main Outcomes and Measures: Prognostic value of TAF for overall survival and disease-specific survival as measured by univariable and multivariable regression analyses. Interpathologist agreement in TAF scoring was also evaluated. Results: A total of 258 colon adenocarcinoma histopathologic cases from 258 patients (138 men [53%]; median age, 67 years [IQR, 65-81 years]) with stage II (n = 119) or stage III (n = 139) cancer were included. Tumor adipose feature was identified in 120 cases (widespread in 63 cases, multifocal in 31, and unifocal in 26). For overall survival analysis after adjustment for tumor stage, TAF was independently prognostic in 2 ways: TAF as a binary feature (presence vs absence: hazard ratio [HR] for presence of TAF, 1.55 [95% CI, 1.07-2.25]; P = .02) and TAF as a semiquantitative categorical feature (HR for widespread TAF, 1.87 [95% CI, 1.23-2.85]; P = .004). Interpathologist agreement for widespread TAF vs lower categories (absent, unifocal, or multifocal) was 90%, corresponding to a κ metric at this threshold of 0.69 (95% CI, 0.58-0.80). Conclusions and Relevance: In this prognostic study, pathologists were able to learn and reproducibly score for TAF, providing significant risk stratification on this independent data set. Although additional work is warranted to understand the biological significance of this feature and to establish broadly reproducible TAF scoring, this work represents the first validation to date of human expert learning from machine learning in pathology. Specifically, this validation demonstrates that a computationally identified histologic feature can represent a human-identifiable, prognostic feature with the potential for integration into pathology practice.

Insights into the Peritumoural Brain Zone of Glioblastoma: CDK4 and EXT2 May Be Potential Drivers of Malignancy.

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.

BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

Co-targeting triple-negative breast cancer cells and endothelial cells by metronomic chemotherapy inhibits cell regrowth and migration via downregulation of the FAK/VEGFR2/VEGF axis and autophagy/apoptosis activation.

High-dose standard-of-care chemotherapy is the only option for triple-negative breast cancer (TNBC) patients, which eventually die due to metastatic tumors. Recently, metronomic chemotherapy (mCHT) showed advantages in treating TNBCs leading us to investigate the anti-metastatic and anti-angiogenic potential of metronomic 5-Fluorouracil plus Vinorelbine (5-FU+VNR) on endothelial cells (ECs) and TNBCs in comparison to standard treatment (STD). We found that 10-fold lower doses of 5-FU+VNR given mCHT vs. STD inhibits cell proliferation and survival of ECs and TNBC cells. Both schedules strongly affect ECs migration and invasion, but in TNBC cells mCHT is significantly more effective than STD in impairing cell migration and invasion. The two treatments disrupt FAK/VEGFR/VEGF signaling in both ECs and TNBC cells. mCHT, and to a much lesser extent STD treatment, induces apoptosis in ECs, whereas it switches the route of cell death from apoptosis (as induced by STD) to autophagy in TNBC cells. mCHT-treated TNBCs-derived conditioned medium also strongly affects ECs' migration, modulates different angiogenesis-associated proteins, and hampers angiogenesis in matrix sponge in vivo. In conclusion, mCHT administration of 5-FU+VNR is more effective than STD schedule in controlling cell proliferation/survival and migration/invasion of both ECs and TNBC cells and has a strong anti-angiogenic effect. Our data suggest that the stabilization of tumor growth observed in TNBC patients treated with mCHT therapy schedule is likely due not only to direct cytotoxic effects but also to anti-metastatic and anti-angiogenic effects.

Paediatric biobanking for health: The ethical, legal, and societal landscape.

Biobanks play a central role in pediatric translational research, which deals primarily with genetic data from sample-based research. However, participation of children in biobanking has received only limited attention in the literature, even though research in general and in clinical trials in particular have a long history in involving minors. So, we resolved to explore specific challenging ethical, legal, and societal issues (ELSI) in the current pediatric biobanking landscape to propose a way forward for biobanking with children as partners in research. Methodologically, we first established the accessibility and utilization of pediatric biobanks, mainly in Europe. This was supported by a literature review related to children's participation, taking into account not only academic papers but also relevant guidelines and best-practices. Our findings are discussed under five themes: general vulnerability; ethical issues-balancing risks and benefits, right to an open future, return of results including secondary findings; legal issues-capacity and legal majority; societal issues-public awareness and empowerment; and responsible research with children. Ultimately, we observed an on-going shift from the parents'/guardians' consent being a sine-qua-non condition to the positive minor's agreement: confirming that the minor is the participant, not the parent(s)/guardian(s). This ethical rethinking is paving the way toward age-appropriate, dynamic and participatory models of involving minors in decision-making. However, we identified a requirement for dynamic tools to assess maturity, a lack of co-produced engagement tools and paucity of shared best practices. We highlight the need to provide empowerment and capability settings to support researchers and biobankers, and back this with practical examples. In conclusion, equipping children and adults with appropriate tools, and ensuring children's participation is at the forefront of responsible pediatric biobanking, is an ethical obligation, and a cornerstone for research integrity.

Exome Sequencing in an ADSHE Family: VUS Identification and Limits.

Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is the familial form of a focal epilepsy characterized by hyperkinetic focal seizures, mainly arising during non-rapid eye movements (NREM) sleep. Mutations associated with ADSHE account for a small proportion of the genetically determined cases, suggesting the existence of other disease-causing genes. Here, we reported the results obtained by performing trio-based whole-exome sequencing (WES) in an Italian family showing ADSHE and investigated the structural impact of putative variants by in silico modeling analysis. We identified a p.(Trp276Gly) variant in MOXD1 gene encoding the monooxigenase DBH like 1 protein, cosegregating with the disease and annotated as VUS under the ACMG recommendations. Structural bioinformatic analysis predicted a high destabilizing effect of this variant, due to the loss of important hydrophilic bonds and an expansion of cavity volume in the protein hydrophobic core. Although our data support a functional effect of the p.(Trp276Gly) variant, we highlight the need to identify additional families carrying MOXD1 mutations or functional analyses in suitable models to clarify its role in ADSHE pathogenesis. Moreover, we discuss the importance of VUS reporting due to the low rate of pathogenic variant identification by NGS in epilepsy and for future reinterpretation studies.

Tumor Microenvironment and Immune Escape in the Time Course of Glioblastoma.

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.

BTK, the new kid on the (oncology) block?

In the last decade data piled up indicating that BTK - for twenty years considered as a "private matter" of bone marrow-derived cells - it is expressed and plays important and different roles also outside of the hematopoietic compartment and, most notably, in tumor cells. Initial evidence that BTK plays a critical role in B cell-derived malignancies prompted the chase for specific inhibitors, the forefather of which entered the clinic in a record time and paved the way for an ever increasing number of new molecules to be trialed. The growing interests in BTK also led to the discovery that, in solid tumors, two novel isoforms are mainly expressed and actionable liabilities for target therapy. Remarkably, the different isoforms appear to be involved in different signaling pathways which will have to be attentively specified in order to define the area of therapeutic intervention. In this perspective we briefly summarize the progress made in the last decade in studying BTK and its isoforms in cancer cells and define the open questions to be addressed in order to get the most benefits from its targeting for therapeutic purposes.

Analysis of copy number alterations in bladder cancer stem cells revealed a prognostic role of LRP1B.

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.