Evaluating cell culture reliability in pediatric brain tumor primary cells through DNA methylation profiling.

In vitro models of pediatric brain tumors (pBT) are instrumental for better understanding the mechanisms contributing to oncogenesis and testing new therapies; thus, ideally, they should recapitulate the original tumor. We applied DNA methylation (DNAm) and copy number variation (CNV) profiling to characterize 241 pBT samples, including 155 tumors and 86 pBT-derived cell cultures, considering serum vs serum-free conditions, late vs early passages, and dimensionality (2D vs 3D cultures). We performed a t-SNE classification and identified differentially methylated regions in tumors compared to cell models. Early cell cultures recapitulate the original tumor, but serum media and 2D culturing were demonstrated to significantly contribute to the divergence of DNAm profiles from the parental ones. All divergent cells clustered together acquiring a common deregulated epigenetic signature suggesting a shared selective pressure. We identified a set of hypomethylated genes shared among unfaithful cells converging on response to growth factors and migration pathways, such as signaling cascade activation, tissue organization, and cellular migration. In conclusion, DNAm and CNV are informative tools that should be used to assess the recapitulation of pBT-cells from parental tumors.

Clinicopathological and molecular landscape of 5-year IDH-wild-type glioblastoma survivors: A multicentric retrospective study.

Five-year glioblastoma (GBM) survivors (LTS) are the minority of the isocitrate dehydrogenase (IDH)-wild-type GBM patients, and their molecular fingerprint is still largely unexplored. This multicenter retrospective study analyzed a large LTS-GBM cohort from nine Italian institutions and molecularly characterized a subgroup of patients by mutation, DNA methylation (DNAm) and copy number variation (CNV) profiling, comparing it to standard survival GBM. Mutation scan allowed the identification of pathogenic variants in most cases, showing a similar mutational spectrum in both groups, and highlighted TP53 as the most commonly mutated gene in the LTS group. We confirmed DNAm as a valuable tool for GBM classification with a diagnostic refinement by using brain tumor classifier v12.5. LTS were more heterogeneous with more cases classified as diffuse pediatric high-grade glioma subtypes and having peculiar CNVs. We observed a global higher methylation in CpG islands and in gene promoters of LTS with methylation levels of distinct gene promoters correlating with prognosis.

Malignant peripheral nerve sheath tumor (MPNST) and MPNST-like entities are defined by a specific DNA methylation profile in pediatric and juvenile population.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) account for 3-10% of pediatric sarcomas, 50% of which occur in neurofibromatosis type 1 (NF1). Sporadic MPNSTs diagnosis may be challenging due to the absence of specific markers, apart from immunohistochemical H3K27me3 loss. DNA methylation (DNAm) profiling is a useful tool for brain and mesenchymal neoplasms categorization, and MPNSTs exhibit a specific DNAm signature. An MPNST-like group has recently been recognized, including pediatric tumors with retained H3K27me3 mark and clinical/histological features not yet well explored. This study aims to characterize the DNAm profile of pediatric/juvenile MPNSTs/MPNST-like entities and its diagnostic/prognostic relevance. RESULTS: We studied 42 tumors from two groups. Group 1 included 32 tumors histologically diagnosed as atypical neurofibroma (ANF) (N = 5) or MPNST (N = 27); group 2 comprised 10 tumors classified as MPNST-like according to Heidelberg sarcoma classifier. We performed further immunohistochemical and molecular tests to reach an integrated diagnosis. In group 1, DNAm profiling was inconclusive for ANF; while, it confirmed the original diagnosis in 12/27 MPNSTs, all occurring in NF1 patients. Five/27 MPNSTs were classified as MPNST-like: Integrated diagnosis confirmed MPNST identity for 3 cases; while, the immunophenotype supported the change to high-grade undifferentiated spindle cell sarcoma in 2 samples. The remaining 10/27 MPNSTs variably classified as schwannoma, osteosarcoma, BCOR-altered sarcoma, rhabdomyosarcoma (RMS)-MYOD1 mutant, RMS-like, and embryonal RMS or did not match with any defined entity. Molecular analysis and histologic review confirmed the diagnoses of BCOR, RMS-MYOD1 mutant, DICER1-syndrome and ERMS. Group 2 samples included 5 high-grade undifferentiated sarcomas/MPNSTs and 5 low-grade mesenchymal neoplasms. Two high-grade and 4 low-grade lesions harbored tyrosine kinase (TRK) gene fusions. By HDBSCAN clustering analysis of the whole cohort we identified two clusters mainly distinguished by H3K27me3 epigenetic signature. Exploring the copy number variation, high-grade tumors showed frequent chromosomal aberrations and CDKN2A/B loss significantly impacted on survival in the MPNSTs cohort. CONCLUSION: DNAm profiling is a useful tool in diagnostic work-up of MPNSTs. Its application in a retrospective series collected during pre-molecular era contributed to classify morphologic mimics. The methylation group MPNST-like is a 'hybrid' category in pediatrics including high-grade and low-grade tumors mainly characterized by TRK alterations.

Patient- and xenograft-derived organoids recapitulate pediatric brain tumor features and patient treatments.

Brain tumors are the leading cause of cancer-related death in children. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of pediatric brain cancers are limited and hard to establish. We present a protocol that enables efficient generation, expansion, and biobanking of pediatric brain cancer organoids. Utilizing our protocol, we have established patient-derived organoids (PDOs) from ependymomas, medulloblastomas, low-grade glial tumors, and patient-derived xenograft organoids (PDXOs) from medulloblastoma xenografts. PDOs and PDXOs recapitulate histological features, DNA methylation profiles, and intratumor heterogeneity of the tumors from which they were derived. We also showed that PDOs can be xenografted. Most interestingly, when subjected to the same routinely applied therapeutic regimens, PDOs respond similarly to the patients. Taken together, our study highlights the potential of PDOs and PDXOs for research and translational applications for personalized medicine.

ß-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum.

Development of the cerebellum is characterized by rapid proliferation of cerebellar granule cell precursors (GCPs) induced by paracrine stimulation of Sonic hedgehog (Shh) signaling from Purkinje cells, in the external granular layer (EGL). Then, granule cell precursors differentiate and migrate into the inner granular layer (IGL) of the cerebellum to form a terminally differentiated cell compartment. Aberrant activation of Sonic hedgehog signaling leads to granule cell precursors hyperproliferation and the onset of Sonic hedgehog medulloblastoma (MB), the most common embryonal brain tumor. ß-arrestin1 (ARRB1) protein plays an important role downstream of Smoothened, a component of the Sonic hedgehog pathway. In the medulloblastoma context, ß-arrestin1 is involved in a regulatory axis in association with the acetyltransferase P300, leading to the acetylated form of the transcription factor E2F1 (E2F1-ac) and redirecting its activity toward pro-apoptotic gene targets. This axis in the granule cell precursors physiological context has not been investigated yet. In this study, we demonstrate that ß-arrestin1 has antiproliferative and pro-apoptotic functions in cerebellar development. ß-arrestin1 silencing increases proliferation of Sonic hedgehog treated-cerebellar precursor cells while decreases the transcription of E2F1-ac pro-apoptotic targets genes, thus impairing apoptosis. Indeed, chromatin immunoprecipitation experiments show a direct interaction between ß-arrestin1 and the promoter regions of the pro-apoptotic E2F1 target gene and P27, indicating the double role of ß-arrestin1 in controlling apoptosis and cell cycle exit in a physiological context. Our data elucidate the role of ß-arrestin1 in the early postnatal stages of cerebellar development, in those cell compartments that give rise to medulloblastoma. This series of experiments suggests that the physiological function of ß-arrestin1 in neuronal progenitors is to directly control, cooperating with E2F1 acetylated form, transcription of pro-apoptotic genes.

Nanoparticles for Drug and Gene Delivery in Pediatric Brain Tumors' Cancer Stem Cells: Current Knowledge and Future Perspectives.

Primary malignant brain tumors are the most common solid neoplasm in childhood. Despite recent advances, many children affected by aggressive or metastatic brain tumors still present poor prognosis, therefore the development of more effective therapies is urgent. Cancer stem cells (CSCs) have been discovered and isolated in both pediatric and adult patients with brain tumors (e.g., medulloblastoma, gliomas and ependymoma). CSCs are a small clonal population of cancer cells responsible for brain tumor initiation, maintenance and progression, displaying resistance to conventional anticancer therapies. CSCs are characterized by a specific repertoire of surface markers and intracellular specific pathways. These unique features of CSCs biology offer the opportunity to build therapeutic approaches to specifically target these cells in the complex tumor bulk. Treatment of pediatric brain tumors with classical chemotherapeutic regimen poses challenges both for tumor location and for the presence of the blood-brain barrier (BBB). Lastly, the application of chemotherapy to a developing brain is followed by long-term sequelae, especially on cognitive abilities. Novel avenues are emerging in the therapeutic panorama taking advantage of nanomedicine. In this review we will summarize nanoparticle-based approaches and the efficacy that NPs have intrinsically demonstrated and how they are also decorated by biomolecules. Furthermore, we propose novel cargoes together with recent advances in nanoparticle design/synthesis with the final aim to specifically target the insidious CSCs population in the tumor bulk.

Modeling Brain Tumors: A Perspective Overview of in vivo and Organoid Models.

Brain tumors are a large and heterogeneous group of neoplasms that affect the central nervous system and include some of the deadliest cancers. Almost all the conventional and new treatments fail to hinder tumoral growth of the most malignant brain tumors. This is due to multiple factors, such as intra-tumor heterogeneity, the microenvironmental properties of the human brain, and the lack of reliable models to test new therapies. Therefore, creating faithful models for each tumor and discovering tailored treatments pose great challenges in the fight against brain cancer. Over the years, different types of models have been generated, and, in this review, we investigated the advantages and disadvantages of the models currently used.

Epigenetic modulators for brain cancer stem cells: Implications for anticancer treatment.

Primary malignant brain tumors are a major cause of morbidity and mortality in both adults and children, with a dismal prognosis despite multimodal therapeutic approaches. In the last years, a specific subpopulation of cells within the tumor bulk, named cancer stem cells (CSCs) or tumor-initiating cells, have been identified in brain tumors as responsible for cancer growth and disease progression. Stemness features of tumor cells strongly affect treatment response, leading to the escape from conventional therapeutic approaches and subsequently causing tumor relapse. Recent research efforts have focused at identifying new therapeutic strategies capable of specifically targeting CSCs in cancers by taking into consideration their complex nature. Aberrant epigenetic machinery plays a key role in the genesis and progression of brain tumors as well as inducing CSC reprogramming and preserving CSC characteristics. Thus, reverting the cancer epigenome can be considered a promising therapeutic strategy. Three main epigenetic mechanisms have been described: DNA methylation, histone modifications, and non-coding RNA, particularly microRNAs. Each of these mechanisms has been proven to be targetable by chemical compounds, known as epigenetic-based drugs or epidrugs, that specifically target epigenetic marks. We review here recent advances in the study of epigenetic modulators promoting and sustaining brain tumor stem-like cells. We focus on their potential role in cancer therapy.

Targeting cancer stem cells in medulloblastoma by inhibiting AMBRA1 dual function in autophagy and STAT3 signalling.

Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBGroup3) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MBGroup3 stem cells, and a combined anti-autophagy and anti-STAT3 approach impacts the MBGroup3 outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.

The role of FOSL1 in stem-like cell reprogramming processes.

Cancer stem-like cells (CSCs) have self-renewal abilities responsible for cancer progression, therapy resistance, and metastatic growth. The glioblastoma stem-like cells are the most studied among CSC populations. A recent study identified four transcription factors (SOX2, SALL2, OLIG2, and POU3F2) as the minimal core sufficient to reprogram differentiated glioblastoma (GBM) cells into stem-like cells. Transcriptomic data of GBM tissues and cell lines from two different datasets were then analyzed by the SWItch Miner (SWIM), a network-based software, and FOSL1 was identified as a putative regulator of the previously identified minimal core. Herein, we selected NTERA-2 and HEK293T cells to perform an in vitro study to investigate the role of FOSL1 in the reprogramming mechanisms. We transfected the two cell lines with a constitutive FOSL1 cDNA plasmid. We demonstrated that FOSL1 directly regulates the four transcription factors binding their promoter regions, is involved in the deregulation of several stemness markers, and reduces the cells' ability to generate aggregates increasing the extracellular matrix component FN1. Although further experiments are necessary, our data suggest that FOSL1 reprograms the stemness by regulating the core of the four transcription factors.

Downregulation of miR-326 and its host gene ß-arrestin1 induces pro-survival activity of E2F1 and promotes medulloblastoma growth.

Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR-326 and its host gene ß-arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro-survival function. Our models revealed that miR-326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation-associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR-326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR-326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro-apoptotic activity. Similar to miR-326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR-326/ARRB1 expression, limiting E2F1 pro-proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression.

Loss of Function SETD2 Mutations in Poorly Differentiated Metastases from Two Hürthle Cell Carcinomas of the Thyroid.

Hürthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability. Functional studies of the novel p.D1890fs6* mutation (case 1) revealed significantly reduced H3K36me3 levels in SETD2-mutated tissue and primary cell cultures and decreased levels of the active form of p53. Restoration of SETD2-wildtype expression in the SETD2-mutant cells significantly reduced the expression of four well-known stemness markers (OCT-4, SOX2, IPF1, Goosecoid). These findings suggest potential roles for SETD2 loss-of-function mutations in HCC progression, possibly involving p53 destabilization and promotion of stemness. Their prevalence and potential treatment implications in thyroid cancer, especially HCC, require further study.

Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue.

PURPOSE: The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources. METHODS: We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay. RESULTS: All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2-5% mutant allele frequency. CONCLUSIONS: This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

Performance of a dual-component molecular assay in cytologically indeterminate thyroid nodules.

PURPOSE: Deciding whether patients with a cytologically indeterminate thyroid nodule should be referred for surgery or for active surveillance is an important challenge for clinicians. The aim of this study was to evaluate the performance of a novel dual-component molecular assay as an ancillary molecular method for resolving indeterminate thyroid nodule cytology. METHODS: We selected 156 thyroid nodules from those that had undergone fine-needle aspiration processed by liquid-based cytology and surgical resection between June 2016 and December 2017. The sample set included 63 nodules cytologically classified as indeterminate, and 93 other nodules randomly selected from those with non-diagnostic, benign, suspicious, or malignant cytology. Nucleic acids from each nodule were subjected to next-generation sequencing analysis for mutation detection in 23 genes and to digital polymerase chain reaction (PCR) evaluation for miR-146b-5p expression levels. RESULTS: Used alone, mutation analysis in the indeterminate subset (cancer prevalence: 22.5%) displayed high sensitivity (89%) and NPV (96%). In contrast, the miR-146b-5p assay offered high specificity (93%) and PPV (93%). Combined use of both analyses improved panel performance by eliminating false-negative results. CONCLUSIONS: These preliminary data suggest that a dual-component molecular test can increase the diagnostic accuracy of thyroid cytology alone by reducing the number of nodules that will be classified as indeterminate and increasing those that can be reliably classified as benign. If these findings are confirmed, this test can be considered for use in clinical practice and is expected to reduce diagnostic surgery and health care costs, and to improve patient quality of life.

Correction to: Exploring the molecular insights of concurrent composite mucoepidermoid carcinoma and papillary thyroid carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Low Doses of Methylmercury Induce the Proliferation of Thyroid Cells In Vitro Through Modulation of ERK Pathway.

Exposure to environmental endocrine disruptors has been associated with an increased frequency of thyroid pathology. In this study, we evaluated the effects of various concentrations of methylmercury (MeHg) on immortalized, non-tumorigenic thyroid cells (Nthy-ori-3-1). Exposure to MeHg at 2.5 and 5 µM for 24 h caused a reduction in cell viability with a decrease of the cell population in sub-G0 phase, as detected by MTT and flow cytometry. Conversely, MeHg at the lower concentration of 0.1 µM increased the cell viability with a rise of G2/M phase. An immunoblot analysis showed higher expression levels of phospho-ERK and not of phospho-Akt. Further enhancement of the cell growth rate was observed after a prolonged exposure of the cells up to 18 days to MeHg 0.1 µM. The present findings demonstrate the toxicity of high concentrations of MeHg on thyroid cells, while showing that treatment with lower doses of Hg, as may occur after prolonged exposure to this environmental contaminant, exerts a promoting effect on thyroid cell proliferation, by acting on the ERK-mediated pro-oncogenic signal transduction pathway.

BRAF mutant colorectal cancer: ErbB2 expression levels as predictive factor for the response to combined BRAF/ErbB inhibitors.

BACKGROUND: Colorectal cancer (CRC) is a heterogeneous disease with a complex biology and a wide number of altered genes such as BRAF, KRAS and PIK3CA. Advances with new-targeted therapies have been achieved and available treating options have prolonged patient's survival. However, BRAF-mutated CRC patients remain unresponsive to available therapies with RAF inhibitors (RAFi) alone or combined with ErbB inhibitors (ErbBi). These unmet needs require further exploitation of oncogenic signaling in order to set up individualized treatments. METHODS: To this end, we tested the efficacy of single agent or combined treatments using the BRAFi, vemurafenib and two different ErbBi: panitumumab and afatinib in CRC cells characterized by different molecular phenotypes. RESULTS: Combination strategies with BRAFi and ErbBi achieved a better response in BRAFV600E mutated cells expressing high levels of ErbB2. CONCLUSIONS: Our findings support the importance of ErbB2 evaluation in BRAF-mutated CRC patients and its role as a positive predictor factor of response to BRAFi/ErbBi combination.

Numb Isoforms Deregulation in Medulloblastoma and Role of p66 Isoform in Cancer and Neural Stem Cells.

Numb is an intracellular protein with multiple functions. The two prevalent isoforms, Numb p66 and Numb p72, are regulators of differentiation and proliferation in neuronal development. Additionally, Numb functions as cell fate determinant of stem cells and cancer stem cells and its abnormal expression has been described in several types of cancer. Involvement of deregulated Numb expression has been described in the malignant childhood brain tumor medulloblastoma, while Numb isoforms in these tumors and in cancer stem-like cells derived from them, have not been studied to date. Here we show that medulloblastoma stem-like cells and cerebellar neuronal stem cells (NSCs) express Numb p66 where its expression tampers stemness features. Furthermore, medulloblastoma samples evaluated in this study express decreased levels of Numb p66 while overexpressed Numb p72 compared with normal tissues. Our results uncover different roles for the two major Numb isoforms examined in medulloblastoma and a critical role for Numb p66 in regulating stem-like cells and NSCs maintenance.