Ultra-Mutation in IDH Wild-Type Glioblastomas of Patients Younger than 55 Years is Associated with Defective Mismatch Repair, Microsatellite Instability, and Giant Cell Enrichment

Glioblastomas (GBMs) are classified into isocitrate dehydrogenase (IDH) mutants and IDH wild-types (IDH-wt). This study aimed at identifying the mutational assets of IDH-wt GBMs in patients aged 18-54 years for which limited data are available

IDH-wild type glioblastomas featuring at least 30% giant cells are characterized by frequent RB1 and NF1 alterations and hypermutation

: Giant cell glioblastoma (GC-GBM) is a rare variant of IDH-wt GBM histologically characterized by the presence of numerous multinucleated giant cells and molecularly considered a hybrid between IDH-wt and IDH-mutant GBM. The lack of an objective definition, specifying the percentage of giant cells required for this diagnosis, may account for the absence of a definite molecular profile of this variant. This study aimed to clarify the molecular landscape of GC-GBM, exploring the mutations and copy number variations of 458 cancer-related genes, tumor mutational burden (TMB), and microsatellite instability (MSI) in 39 GBMs dichotomized into having 30-49% (15 cases) or\u2009 65\u200950% (24 cases) GCs. The type and prevalence of the genetic alterations in this series was not associated with the GCs content (<\u200950% or 65\u200950%). Most cases (82% and 51.2%) had impairment in TP53/MDM2 and PTEN/PI3K pathways, but a high proportion also featured TERT promoter mutations (61.5%) and RB1 (25.6%) or NF1 (25.6%) alterations. EGFR amplification was detected in 18% cases in association with a shorter overall survival (P\u2009=\u20090.004). Sixteen (41%) cases had a TMB\u2009>\u200910 mut/Mb, including two (5%) that harbored MSI and one with a POLE mutation. The frequency of RB1 and NF1 alterations and TMB counts were significantly higher compared to 567 IDH wild type (P\u2009<\u20090.0001; P\u2009=\u20090.0003; P\u2009<\u20090.0001) and 26 IDH-mutant (P\u2009<\u20090.0001; P\u2009=\u20090.0227; P\u2009<\u20090.0001) GBMs in the TCGA PanCancer Atlas cohort. These findings demonstrate that the molecular landscape of GBMs with at least 30% giant cells is dominated by the impairment of TP53/MDM2 and PTEN/PI3K pathways, and additionally characterized by frequent RB1 alterations and hypermutation and by EGFR amplification in more aggressive cases. The high frequency of hypermutated cases suggests that GC-GBMs might be candidates for immune check-point inhibitors clinical trials

Lack of association of CD44-rs353630 and CHI3L2-rs684559 with pancreatic ductal adenocarcinoma survival.

Although pancreatic ductal adenocarcinoma (PDAC) survival is poor, there are differences in patients' response to the treatments. Detection of predictive biomarkers explaining these differences is of the utmost importance. In a recent study two genetic markers (CD44-rs353630 and CHI3L2-rs684559) were reported to be associated with survival after PDAC resection. We attempted to replicate the associations in 1856 PDAC patients (685 resected with stage I/II) from the PANcreatic Disease ReseArch (PANDoRA) consortium. We also analysed the combined effect of the two genotypes in order to compare our results with what was previously reported. Additional stratified analyses considering TNM stage of the disease and whether the patients received surgery were also performed. We observed no statistically significant associations, except for the heterozygous carriers of CD44-rs353630, who were associated with worse OS (HR = 5.01; 95% CI 1.58-15.88; p = 0.006) among patients with stage I disease. This association is in the opposite direction of those reported previously, suggesting that data obtained in such small subgroups are hardly replicable and should be considered cautiously. The two polymorphisms combined did not show any statistically significant association. Our results suggest that the effect of CD44-rs353630 and CHI3L2-rs684559 cannot be generalized to all PDAC patients

DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs

Regolazione della proteina AKT da parte del complesso PML-RAR attraverso l’inibizione di HSP90 (Heat Shock Protein 90 kD) nella Leucemia Acuta Promielocitica t(15;17)

La proteina HSP90 è uno chaperone molecolare necessario per l’attivazione e la stabilizzazione di numerose proteine coinvolte nel ciclo cellulare, nella trasduzione del segnale, nel processo apoptotico e con funzione recettoriale. Tale chaperone è presente in quantità elevata in tutte le cellule, ma è differenzialmente espresso nei diversi tessuti durante lo sviluppo embrionale e nelle cellule staminali. HSP90 svolge la sua funzione in forma di omodimero, i cui due monomeri (isoforma alpha () e beta (sono costituiti da tre principali domini: un dominio N-terminale con attività ATP-dipendente, un dominio intermedio e un dominio di dimerizzazione. La forma HSP90 beta è ubiquitaria, a differenza dell’isoforma alpha che aumenta invece in condizioni di stress, in molti tumori e nelle cellule staminali. Le isoforme alpha e beta sono regolate ciascuna dal proprio promotore e la loro espressione e’ in stretto equilibrio. In pazienti con leucemia acuta mieloide (LMA), livelli elevati della proteina HSP90 correlano con una prognosi infausta per il paziente. In questo studio abbiamo dimostrato che l’espressione della proteina HSP90 viene inibita a livello trascrizionale e traslazionale in cellule di pazienti con leucemia acuta promielocitica (LAP). La LAP è un sottotipo distinto di LMA, caratterizzato dalla presenza della traslocazione t(15;17), che da’ origine alla proteina di fusione PML-RAR in grado di inibire l’espressione di vari geni coinvolti nel differenziamento cellulare e nel apoptosi. I blasti della LAP sono sensibili all’azione dell’acido all-trans-retinoico (ATRA) ed al triossido di arsenico (ATO), molecole in grado di indurre il differenziamento cellulare. La serina-treonina chinasi AKT svolge un ruolo regolatore cruciale in differenti processi biologici quali differenziamento cellulare, ciclo cellulare, trascrizione, traduzione, metabolismo e apoptosi, ed è una proteina regolata da HSP90. Abbiamo 9 dimostrato che nella linea cellulare promielocitica NB4, l’attività nucleare della proteina AKT aumenta durante il differenziamento indotto dall’ATRA. Inoltre, abbiamo osservato come il trattamento in-vitro di cellule PML-RAR positive (NB4, PR9 e blasti leucemici primari) con ATRA e ATO siano in grado di ristabilire una corretta espressione di HSP90. Effettuando esperimenti di immunoprecipitazione della cromatina abbiamo dimostrato come PML-RAR interagisca direttamente con i promotori delle isoforme alpha e beta di HSP90 e come il trattamento delle cellule NB4 con ATRA aumenti l’acetilazione delle due isoforme . Abbiamo quindi dimostrato che la presenza di PML-RAR inibisce l'espressione di HSP90 a livello trascrizionale attraverso il reclutamento del complesso repressore NCORHDAC nel promotore dei geni che codificano per le isoforme alpha e beta. Abbiamo inoltre evidenziato la down-regolazione della proteina AKT nei pazienti con leucemia acuta promielocitica e abbiamo osservato come PML-RAR regoli negativamente l'espressione post-traduzionale di AKT attraverso la down-regolazione di HSP90.HSP90 is a molecular chaperone required for activation and stabilization of numerous proteins involved in cell cycling, receptor function, signal transduction and apoptosis pathways. HSP90 is abundant in all cells and is differentially expressed in several tissues during the embryonic development and in stem cells. HSP90 functions as a homodimer with each monomer consisting of three major domains: the N-terminal ATP-domain, the middle domain and the Cterminal dimerization domain. The two major isoforms of this molecular chaperone include HSP90 alpha and beta. The HSP90 beta isoform is ubiquitously highly expressed, whereas the HSP90 alpha isoform is stress-inducible and is over-expressed in many tumors and in stem cells. Both HSP90 alpha and beta are regulated by specific promoters and their reciprocal expression is balanced and is tightly controlled. In patients with acute myeloid leukemia (AML), higher levels of the HSP90 protein have been associated with poor prognosis. Here, we report transcriptional and translational inhibition of HSP90s in primary blasts from patients with acute promyelocytic leukemia (APL). APL is a unique subtype of AML characterized by the presence of the t(15;17) translocation, giving rise to the PML-RAR fusion protein that deregulates the expression of various genes involved in differentiation and apoptosis pathways. APL cells have been shown to be sensitive to all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). The serine - threonine kinase AKT, plays crucial regulatory roles in different biological processes such as cell differentiation, cell cycle, transcription, translation, metabolism and apoptosis and is a client protein of HSP90. We found that nuclear AKT activity increases during ATRAmediated differentiation of the human APL cell line (NB4). Furthermore, we were able to show that in-vitro treatment of PML-RAR expressing cells (NB4 cells, PR9 cells and primary APL blasts) with ATRA and ATO restores the balanced expression of HSP90. Using chromatin immunoprecipitation assays, we demonstrated that PML-RAR binds to the HSP90 alpha and beta promoter regions, and that treatment of NB4 cells with ATRA induces promoter acetylation of the two HSP90 isoforms. Our study shows for the first time that the presence of the PML-RAR fusion protein inhibits HSP90s expression at the transcriptional level through recruitment of the HDACrepressor complex NCOR in the promoters of HSP90 alpha and beta isoforms. In this line, AKT resulted down-regulated at the protein level in primary APL blasts, where we also demonstrated that PML-RAR negatively regulates the post translational expression of AKT through transcriptional down regulation of HSP90

Regolazione della proteina AKT da parte del complesso PML-RARα attraverso l’inibizione di HSP90 (Heat Shock Protein 90 kD) nella Leucemia Acuta Promielocitica t(15;17)

La proteina HSP90 è uno chaperone molecolare necessario per l’attivazione e la stabilizzazione di numerose proteine coinvolte nel ciclo cellulare, nella trasduzione del segnale, nel processo apoptotico e con funzione recettoriale. Tale chaperone è presente in quantità elevata in tutte le cellule, ma è differenzialmente espresso nei diversi tessuti durante lo sviluppo embrionale e nelle cellule staminali. HSP90 svolge la sua funzione in forma di omodimero, i cui due monomeri (isoforma alpha () e beta (sono costituiti da tre principali domini: un dominio N-terminale con attività ATP-dipendente, un dominio intermedio e un dominio di dimerizzazione. La forma HSP90 beta è ubiquitaria, a differenza dell’isoforma alpha che aumenta invece in condizioni di stress, in molti tumori e nelle cellule staminali. Le isoforme alpha e beta sono regolate ciascuna dal proprio promotore e la loro espressione e’ in stretto equilibrio. In pazienti con leucemia acuta mieloide (LMA), livelli elevati della proteina HSP90 correlano con una prognosi infausta per il paziente. In questo studio abbiamo dimostrato che l’espressione della proteina HSP90 viene inibita a livello trascrizionale e traslazionale in cellule di pazienti con leucemia acuta promielocitica (LAP). La LAP è un sottotipo distinto di LMA, caratterizzato dalla presenza della traslocazione t(15;17), che da’ origine alla proteina di fusione PML-RAR in grado di inibire l’espressione di vari geni coinvolti nel differenziamento cellulare e nel apoptosi. I blasti della LAP sono sensibili all’azione dell’acido all-trans-retinoico (ATRA) ed al triossido di arsenico (ATO), molecole in grado di indurre il differenziamento cellulare. La serina-treonina chinasi AKT svolge un ruolo regolatore cruciale in differenti processi biologici quali differenziamento cellulare, ciclo cellulare, trascrizione, traduzione, metabolismo e apoptosi, ed è una proteina regolata da HSP90. Abbiamo dimostrato che nella linea cellulare promielocitica NB4, l’attività nucleare della proteina AKT aumenta durante il differenziamento indotto dall’ATRA. Inoltre, abbiamo osservato come il trattamento in-vitro di cellule PML-RAR positive (NB4, PR9 e blasti leucemici primari) con ATRA e ATO siano in grado di ristabilire una corretta espressione di HSP90. Effettuando esperimenti di immunoprecipitazione della cromatina abbiamo dimostrato come PML-RAR interagisca direttamente con i promotori delle isoforme alpha e beta di HSP90 e come il trattamento delle cellule NB4 con ATRA aumenti l’acetilazione delle due isoforme . Abbiamo quindi dimostrato che la presenza di PML-RAR inibisce l'espressione di HSP90 a livello trascrizionale attraverso il reclutamento del complesso repressore NCORHDAC nel promotore dei geni che codificano per le isoforme alpha e beta. Abbiamo inoltre evidenziato la down-regolazione della proteina AKT nei pazienti con leucemia acuta promielocitica e abbiamo osservato come PML-RAR regoli negativamente l'espressione post-traduzionale di AKT attraverso la down-regolazione di HSP90.HSP90 is a molecular chaperone required for activation and stabilization of numerous proteins involved in cell cycling, receptor function, signal transduction and apoptosis pathways. HSP90 is abundant in all cells and is differentially expressed in several tissues during the embryonic development and in stem cells. HSP90 functions as a homodimer with each monomer consisting of three major domains: the N-terminal ATP-domain, the middle domain and the Cterminal dimerization domain. The two major isoforms of this molecular chaperone include HSP90 alpha and beta. The HSP90 beta isoform is ubiquitously highly expressed, whereas the HSP90 alpha isoform is stress-inducible and is over-expressed in many tumors and in stem cells. Both HSP90 alpha and beta are regulated by specific promoters and their reciprocal expression is balanced and is tightly controlled. In patients with acute myeloid leukemia (AML), higher levels of the HSP90 protein have been associated with poor prognosis. Here, we report transcriptional and translational inhibition of HSP90s in primary blasts from patients with acute promyelocytic leukemia (APL). APL is a unique subtype of AML characterized by the presence of the t(15;17) translocation, giving rise to the PML-RAR fusion protein that deregulates the expression of various genes involved in differentiation and apoptosis pathways. APL cells have been shown to be sensitive to all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). The serine - threonine kinase AKT, plays crucial regulatory roles in different biological processes such as cell differentiation, cell cycle, transcription, translation, metabolism and apoptosis and is a client protein of HSP90. We found that nuclear AKT activity increases during ATRAmediated differentiation of the human APL cell line (NB4). Furthermore, we were able to show that in-vitro treatment of PML-RAR expressing cells (NB4 cells, PR9 cells and primary APL blasts) with ATRA and ATO restores the balanced expression of HSP90. Using chromatin immunoprecipitation assays, we demonstrated that PML-RAR binds to the HSP90 alpha and beta promoter regions, and that treatment of NB4 cells with ATRA induces promoter acetylation of the two HSP90 isoforms. Our study shows for the first time that the presence of the PML-RAR fusion protein inhibits HSP90s expression at the transcriptional level through recruitment of the HDACrepressor complex NCOR in the promoters of HSP90 alpha and beta isoforms. In this line, AKT resulted down-regulated at the protein level in primary APL blasts, where we also demonstrated that PML-RAR negatively regulates the post translational expression of AKT through transcriptional down regulation of HSP90

Recurrent oligodendroglioma with changed 1p/19q status

: We report a case of oligodendroglioma that had consistent histopathological features as well as a distinct change in 1p/19q status in the second recurrence, after temozolomide chemotherapy and radiotherapy. The first tumor recurrence had oligodendroglial morphology, IDH1 R132H and TERT promoter mutations, and 1p/19q codeletion detected by fluorescent in situ hybridization (FISH). Copy number analysis, assessed by next-generation sequencing, confirmed 1p/19q codeletion, and disclosed loss of heterozygosity (LOH) of chromosomes 4 and 9 and chromosome 11 gain. The second recurrence featured not only oligodendroglial morphology but also the appearance of admixed multinucleated giant cells or neoplastic cells having oval nuclei and mitoses and showing microvascular proliferation; it maintained IDH1 R132H and TERT promoter mutations, acquired TP53 mutation, and showed 19q LOH, but disomic 1p, detected by FISH. Copy number analysis depicted LOH of chromosomes 3p, 13, and 19q, 1p partial deletion (1p chr1p34.2-p11), and gain of chromosomes 2p25.3-p24.1, 8q12.2-q24.3, and 11q13.3-q25. B-allele frequency analysis of polymorphic sites disclosed copy-neutral LOH at 1p36.33-p34.2, supporting the initial deletion of 1p, followed by reduplication of 1p36.33-p34.2 alone. These findings suggest that the two tumor recurrences might have originated from an initial neoplastic clone, featuring 1p/19q codeletion and IDH1 and TERT promoter mutations, and have independently acquired other copy number alterations. The reduplication of chromosome 1p might be the result of temozolomide treatment, and gave rise to false negative 1p deletion detected by FISH. The possibility of 1p copy-neutral LOH should be considered in recurrent oligodendrogliomas with altered 1p/19q status detected by FISH

Molecular Profiling of 22 Primary Atypical Meningiomas Shows the Prognostic Significance of 18q Heterozygous Loss and CDKN2A/B Homozygous Deletion on Recurrence-Free Survival

The use of adjuvant therapy is controversial in atypical meningiomas with gross total resection. Predictors of recurrence risk could be useful in selecting patients for additional treatments. The aim of this study was to investigate whether molecular features are associated with recurrence risk of atypical meningiomas. According to WHO classification, the diagnosis of atypical meningioma was based on the presence of one major criteria (mitotic activity, brain invasion) or three or more minor criteria. The molecular profile of 22 cases (eight mitotically active, eight brain-invasive, and six with minor criteria) was assessed exploring the mutational status and copy number variation of 409 genes using next generation sequencing. Of the 22 patients with a median follow up of 53.5 months, 13 had recurrence of disease within 68 months. NF2 mutation was the only recurrent alteration (11/22) and was unrelated to clinical-pathological features. Recurring meningiomas featured a significantly higher proportion of copy number losses than non-recurring ones (p = 0.027). Chromosome 18q heterozygous loss or CDKN2A/B homozygous deletion was significantly associated with shorter recurrence-free survival (p = 0.008; hazard ratio: 5.3). Atypical meningiomas could be tested routinely for these genetic alterations to identify cases for adjuvant treatment

Poorly Cohesive Gastric Cancers Showing the Transcriptomic Hallmarks of Epithelial-Mesenchymal Transition Behave Aggressively

Poorly cohesive (PC) gastric cancer (GC) exhibits variable clinical behaviour, being extremely aggressive in most cases but more indolent at times. We hypothesized that the integrative genomic and gene expression characterization of a PC GC series could help identifying molecular subtypes with potential clinical implications