A personalized medicine approach identifies enasidenib as an efficient treatment for IDH2 mutant chondrosarcoma.

BACKGROUND: Sarcomas represent an extensive group of malignant diseases affecting mesodermal tissues. Among sarcomas, the clinical management of chondrosarcomas remains a complex challenge, as high-grade tumours do not respond to current therapies. Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes are among the most common mutations detected in chondrosarcomas and may represent a therapeutic opportunity. The presence of mutated IDH (mIDH) enzymes results in the accumulation of the oncometabolite 2-HG leading to molecular alterations that contribute to drive tumour growth. METHODS: We developed a personalized medicine strategy based on the targeted NGS/Sanger sequencing of sarcoma samples (n = 6) and the use of matched patient-derived cell lines as a drug-testing platform. The anti-tumour potential of IDH mutations found in two chondrosarcoma cases was analysed in vitro, in vivo and molecularly (transcriptomic and DNA methylation analyses). FINDINGS: We treated several chondrosarcoma models with specific mIDH1/2 inhibitors. Among these treatments, only the mIDH2 inhibitor enasidenib was able to decrease 2-HG levels and efficiently reduce the viability of mIDH2 chondrosarcoma cells. Importantly, oral administration of enasidenib in xenografted mice resulted in a complete abrogation of tumour growth. Enasidenib induced a profound remodelling of the transcriptomic landscape not associated to changes in the 5 mC methylation levels and its anti-tumour effects were associated with the repression of proliferative pathways such as those controlled by E2F factors. INTERPRETATION: Overall, this work provides preclinical evidence for the use of enasidenib to treat mIDH2 chondrosarcomas. FUNDING: Supported by the Spanish Research Agency/FEDER (grants PID2022-142020OB-I00; PID2019-106666RB-I00), the ISC III/FEDER (PI20CIII/00020; DTS18CIII/00005; CB16/12/00390; CB06/07/1009; CB19/07/00057); the GEIS group (GEIS-62); and the PCTI (Asturias)/FEDER (IDI/2021/000027).

Analysis of ATG4C function in vivo.

ABBREVIATIONS: ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).

Pseudohypoxia in paraganglioma and pheochromocytoma is associated with an immunosuppressive phenotype.

Metastatic pheochromocytoma and paraganglioma (PPGL) have poor prognosis and limited therapeutic options. The recent advent of immunotherapies showing remarkable clinical efficacies against various cancer types offers the possibility of novel opportunities also for metastatic PPGL. Most PPGLs are pathogenically linked to inactivating mutations in genes encoding different succinate dehydrogenase (SDH) subunits. This causes activation of the hypoxia-inducible factor 2 (HIF2)-mediated transcriptional program in the absence of decreased intratumoral oxygen levels, a phenomenon known as pseudohypoxia. Genuine hypoxia in a tumor creates an immunosuppressive tumor microenvironment. However, the impact of pseudohypoxia in the immune landscape of tumors remains largely unexplored. In this study, tumoral expression of programmed death-ligand 1 (PD-L1) and HIF2α and tumor infiltration of CD8 T lymphocytes (CTLs) were examined in PPGL specimens from 102 patients. We assessed associations between PD-L1, CTL infiltration, HIF2α expression, and the mutational status of SDH genes. Our results show that high PD-L1 expression levels in tumor cells and CTL tumor infiltration were more frequent in metastatic than nonmetastatic PPGL. However, this phenotype was negatively associated with SDH mutations and high HIF2α protein expression. These data were validated by analysis of mRNA levels of genes expressing PD-L1, CD8, and HIF2α in PPGL included in The Cancer Genome Atlas database. Further, PD-L1 and CD8 expression was lower in norepinephrine than epinephrine-secreting PPGL. This in silico analysis also revealed the low PD-L1 or CD8 expression levels in tumors with inactivating mutations in VHL or activating mutations in the HIF2α-coding gene, EPAS1, which, together with SDH-mutated tumors, comprise the pseudohypoxic molecular subtype of PPGL. These findings suggest that pseudohypoxic tumor cells induce extrinsic signaling toward the immune cells promoting the development of an immunosuppressive environment. It also provides compelling support to explore the differential response of metastatic PPGL to immune checkpoint inhibitors. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Oncogenic FOXL2 C134W Mutation Is a Key Driver of Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCT) are the most common type of malignant ovarian sex cord-stromal tumors. Most AGCTs carry the somatic variant c.402C>G (p.C134W) affecting the transcription factor FOXL2. Germline dominant variants in FOXL2 are responsible for blepharophimosis syndrome, which is characterized by underdevelopment of the eyelid. In this work, we generated a mouse model harboring the C134W variant of FOXL2 to evaluate in vivo the poorly understood oncogenic role of FOXL2. The mutation was dominant regarding eyelid hypoplasia, reminiscent of blepharophimosis syndrome. Interestingly, Foxl2+/C134W female mice had reduced fertility and developed AGCTs through a progression from abnormal ovaries with aberrant granulosa cells to ovaries with stromal hyperplasia and atypia and on to tumors in adut mice. The genes dysregulated in mouse AGCTs exhibited the hallmarks of cancer and were consistent with a gain-of-function of the mutated allele affecting TGFß signaling. A comparison of these data with previous results on human AGCTs indicated similar deregulated pathways. Finally, a mutational analysis of mouse AGCT transcriptomic data suggested the absence of additional driver mutations apart from FOXL2-C134W. These results provide a clear in vivo example in which a single mutational hit triggers tumor development associated with profound transcriptomic alterations. SIGNIFICANCE: A newly generated mouse model carrying a FOXL2 mutation characteristic of adult-type granulosa cell tumors shows that FOXL2 C134W shifts the transcriptome towards a signature of granulosa cell cancer and drives tumorigenesis.

Tumor-Intrinsic Nuclear ß-Catenin Associates with an Immune Ignorance Phenotype and a Poorer Prognosis in Head and Neck Squamous Cell Carcinomas.

Activation of WNT/ß-catenin signaling has been associated with a non-T-cell-inflamed tumor microenvironment (TME) in several cancers. The aim of this work was to investigate the relationship between ß-catenin signaling and TME inflammation in head and neck squamous cell carcinomas (HNSCCs). Membrane and nuclear ß-catenin expression, PD-L1 expression, and CD8+ tumor-infiltrating lymphocyte (TIL) density were jointly evaluated by immunohistochemistry in a series of 372 HPV-negative HNSCCs. Membrane ß-catenin levels decreased in carcinomas compared to the normal epithelium. Positive nuclear ß-catenin was detected in 50 tumors (14.3%) and was significantly associated with a low CD8+ TIL density (168 cells/mm2 versus 293 cells/mm2 in nuclear-ß-catenin-negative cases; p = 0.01) and a tendency for a lower expression of PD-L1, resulting in association with a noninflamed TME (i.e., type II, immunological ignorance). Multivariate Cox analysis further demonstrated that low infiltration by CD8+ TILs (HR = 1.6, 95% CI = 1.19-2.14, p = 0.002) and nuclear ß-catenin expression (HR = 1.47, 95% CI = 1.01-2.16, p = 0.04) were both independently associated with a poorer disease-specific survival. In conclusion, tumor-intrinsic nuclear ß-catenin activation is associated with a non-inflamed TME phenotype and a poorer prognosis, thereby suggesting a possible implication as an immune exclusion mechanism for a subset of HNSCC patients.

Classification of follicular-patterned thyroid lesions using a minimal set of epigenetic biomarkers.

Objective: The minimally invasive fine-needle aspiration cytology (FNAC) is the current gold standard for the diagnosis of thyroid nodule malignancy. However, the correct discrimination of follicular neoplasia often requires more invasive diagnostic techniques. The lack of suitable immunohistochemical markers to distinguish between follicular thyroid carcinoma and other types of follicular-derived lesions complicates diagnosis, and despite most of these tumours being surgically resected, only a small number will test positive for malignancy. As such, the development of new orthogonal diagnostic approaches may improve the accuracy of diagnosing thyroid nodules. Design: This study includes a retrospective, multi-centre training cohort including 54 fresh-frozen follicular-patterned thyroid samples and two independent, multi-centre validation cohorts of 103 snap-frozen biopsies and 33 FNAC samples, respectively. Methods: We performed a genome-wide genetic and epigenetic profiling of 54 fresh-frozen follicular-patterned thyroid samples using exome sequencing and the Illumina Human DNA Methylation EPIC platform. An extensive validation was performed using the bisulfite pyrosequencing technique. Results: Using a random forest approach, we developed a three-CpG marker-based diagnostic model that was subsequently validated using bisulfite pyrosequencing experiments. According to the validation cohort, this cost-effective method discriminates between benign and malignant nodules with a sensitivity and specificity of 97 and 88%, respectively (positive predictive value (PPV): 0.85, negative predictive value (NPV): 0.98). Conclusions: Our classification system based on a minimal set of epigenetic biomarkers can complement the potential of the diagnostic techniques currently available and would prioritize a considerable number of surgical interventions that are often performed due to uncertain cytology. Significance statement: In recent years, there has been a significant increase in the number of people diagnosed with thyroid nodules. The current challenge is their etiological diagnosis to discount malignancy without resorting to thyroidectomy. The method proposed here, based on DNA pyrosequencing assays, has high sensitivity (0.97) and specificity (0.88) for the identification of malignant thyroid nodules. This simple and cost-effective approach can complement expert pathologist evaluation to prioritize the classification of difficult-to-diagnose follicular-patterned thyroid lesions and track tumor evolution, including real-time monitoring of treatment efficacy, thereby stimulating adherence to health promotion programs.

Differential HIF2α Protein Expression in Human Carotid Body and Adrenal Medulla under Physiologic and Tumorigenic Conditions.

Hypoxia-inducible factors (HIF) 2α and 1α are the major oxygen-sensing molecules in eukaryotic cells. HIF2α has been pathogenically linked to paraganglioma and pheochromocytoma (PPGL) arising in sympathetic paraganglia or the adrenal medulla (AM), respectively. However, its involvement in the pathogenesis of paraganglioma arising in the carotid body (CB) or other parasympathetic ganglia in the head and neck (HNPGL) remains to be defined. Here, we retrospectively analyzed HIF2α by immunohistochemistry in 62 PPGL/HNPGL and human CB and AM, and comprehensively evaluated the HIF-related transcriptome of 202 published PPGL/HNPGL. We report that HIF2α is barely detected in the AM, but accumulates at high levels in PPGL, mostly (but not exclusively) in those with loss-of-function mutations in VHL and genes encoding components of the succinate dehydrogenase (SDH) complex. This is associated with upregulation of EPAS1 and the HIF2α-regulated genes COX4I2 and ADORA2A. In contrast, HIF2α and HIF2α-regulated genes are highly expressed in CB and HNPGL, irrespective of VHL and SDH dysfunctions. We also found that HIF2α and HIF1α protein expressions are not correlated in PPGL nor HNPGL. In addition, HIF1α-target genes are almost exclusively overexpressed in VHL-mutated HNPGL/PPGL. Collectively, the data suggest that involvement of HIF2α in the physiology and tumor pathology of human paraganglia is organ-of-origin-dependent and HIF1α-independent.

A truncating variant of RAD51B associated with primary ovarian insufficiency provides insights into its meiotic and somatic functions.

Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although its genetic etiology has been extensively investigated, most POI cases remain unexplained. Using whole-exome sequencing, we identified a homozygous variant in RAD51B -(c.92delT) in two sisters with POI. In vitro studies revealed that this variant leads to translation reinitiation at methionine 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, Rad51b-c.92delT variant reduced replication fork progression of patient-derived lymphoblastoid cell lines and pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability.

Mechanical ventilation promotes lung tumour spread by modulation of cholesterol cell content.

BACKGROUND: Mechanical stretch of cancer cells can alter their invasiveness. During mechanical ventilation, lungs may be exposed to an increased amount of stretch, but the consequences on lung tumours have not been explored. METHODS: To characterise the influence of mechanical ventilation on the behaviour of lung tumours, invasiveness assays and transcriptomic analyses were performed in cancer cell lines cultured in static conditions or under cyclic stretch. Mice harbouring lung melanoma implants were submitted to mechanical ventilation and metastatic spread was assessed. Additional in vivo experiments were performed to determine the mechanodependent specificity of the response. Incidence of metastases was studied in a cohort of lung cancer patients that received mechanical ventilation compared with a matched group of nonventilated patients. RESULTS: Stretch increases invasiveness in melanoma B16F10luc2 and lung adenocarcinoma A549 cells. We identified a mechanosensitive upregulation of pathways involved in cholesterol processing in vitro, leading to an increase in pro-protein convertase subtilisin/kexin type 9 (PCSK9) and LDLR expression, a decrease in intracellular cholesterol and preservation of cell stiffness. A course of mechanical ventilation in mice harbouring melanoma implants increased brain and kidney metastases 2 weeks later. Blockade of PCSK9 using a monoclonal antibody increased cell cholesterol and stiffness and decreased cell invasiveness in vitro and metastasis in vivo. In patients, mechanical ventilation increased PCSK9 abundance in lung tumours and the incidence of metastasis, thus decreasing survival. CONCLUSIONS: Our results suggest that mechanical stretch promote invasiveness of cancer cells, which may have clinically relevant consequences. Pharmacological manipulation of cholesterol endocytosis could be a novel therapeutic target in this setting.

Epigenetic Deregulation of the Histone Methyltransferase KMT5B Contributes to Malignant Transformation in Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and aggressive type of brain tumor in adulthood. Epigenetic mechanisms are known to play a key role in GBM although the involvement of histone methyltransferase KMT5B and its mark H4K20me2 has remained largely unexplored. The present study shows that DNA hypermethylation and loss of DNA hydroxymethylation is associated with KMT5B downregulation and genome-wide reduction of H4K20me2 levels in a set of human GBM samples and cell lines as compared with non-tumoral specimens. Ectopic overexpression of KMT5B induced tumor suppressor-like features in vitro and in a mouse tumor xenograft model, as well as changes in the expression of several glioblastoma-related genes. H4K20me2 enrichment was found immediately upstream of the promoter regions of a subset of deregulated genes, thus suggesting a possible role for KMT5B in GBM through the epigenetic modulation of key target cancer genes.

Immune Cell Infiltrates and Neutrophil-to-Lymphocyte Ratio in Relation to Response to Chemotherapy and Prognosis in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas.

Our goal was to assess the correlation of immune parameters with the response to induction chemotherapy (ICT) in head and neck squamous cell carcinoma (HNSCC) patients. Pretreatment biopsies from 64 patients with HNSCC that received ICT were assessed for PD-L1 protein expression and density of CD8+ and FOXP3+ tumor infiltrating lymphocytes (TIL). In addition, the neutrophil-to-lymphocyte ratio (NLR) was calculated from pretreatment whole blood counts. In total, 55% of cases exhibited PD-L1 combined proportion score (CPS) positivity (≥1% stained cells). PD-L1 CPS positivity correlated with a high density of both CD8+ (p = 0.01) and FOXP3+ (p < 0.001) TILs. There was no correlation between PD-L1 expression or TIL density and NLR values. In univariate analyses, the absence of PD-L1 CPS expression (p = 0.042) and a high NLR (p = 0.034) were significantly correlated with response to ICT. Neither CD8+ TIL (p = 0.99) nor FOXP3+ TIL densities (p = 0.71) were associated with response to ICT. In multivariate analysis, only a high NLR was associated with response to ICT (HR = 4.06, 95% CI = 1.06-15.5, p = 0.04). In addition, a high NLR was also independently associated with lower disease-specific (p = 0.03) and overall survival rates (p = 0.04), particularly in the subset of patients who received definitive surgical treatment. These results suggest that NLR could emerge as a predictive biomarker of response to ICT.

ARID2 deficiency promotes tumor progression and is associated with higher sensitivity to chemotherapy in lung cancer.

The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.

Impact of notch signaling on the prognosis of patients with head and neck squamous cell carcinoma.

OBJECTIVES: The function of NOTCH signaling (oncogenic or oncosuppressive) remains controversial in head and neck squamous cell carcinomas (HNSCC). The purpose of this work is to investigate the role of NOTCH pathway in HNSCC prognosis. METHODS: Immunohistochemical NOTCH1 and HES1 expression was jointly evaluated and correlated with other NOTCH1 targets, p21 (WAF1/Cip1) and Cyclin D1, using an unbiased cohort of 372 surgically treated HPV-negative HNSCC patients. RESULTS: Membranous NOTCH1 expression was detected in 197 (61%) out of 324 evaluable tumor samples, and nuclear NOTCH1 expression in 91 samples (28%). Nuclear HES1 expression was found in 224 (67%) cases. Membranous and nuclear NOTCH1 expression were consistently and significantly correlated with nuclear HES1 (P < 0.001) and p21 (P = 0.03) expression, but not with Cyclin D1. NOTCH1 expression was significantly associated to early stages (I-II), non-recurrent disease, and better disease-specific (DSS) and overall survival (OS) rates (P < 0.001). Moreover, triple-positive cases (NOTCH1+/HES1+/p21+) exhibited significantly improved DSS (P < 0.001) and OS (P = 0.004), thus reinforcing the association of NOTCH pathway activation with a better prognosis in HNSCC. Multivariate analysis further revealed membranous NOTCH1 expression as a robust independent predictor of better DSS (HR = 0.554; 95% IC 0.412-0.745; P < 0.001) and better OS (HR = 0.640; 95% CI 0.491-0.835; P = 0.001). CONCLUSION: These findings show the association of NOTCH pathway activation with a better prognosis in HNSCC patients, also revealing membranous NOTCH1 expression as a robust independent predictor of improved survival. Accordingly, these results suggest a tumor suppressive rather than an oncogenic role for NOTCH pathway in HNSCC.

Characterization of Left Ventricular Non-Compaction Cardiomyopathy.

Left ventricle non-compaction cardiomyopathy (LVNC) has gained great interest in recent years, being one of the most controversial cardiomyopathies. There are several open debates, not only about its genetic heterogeneity, or about the possibility to be an acquired cardiomyopathy, but also about its possible overdiagnosis based on imaging techniques. In order to better understand this entity, we identified 38 LVNC patients diagnosed by cardiac MRI (CMRI) or anatomopathological study that could underwent NGS-sequencing and clinical study. Anatomopathological exam was performed in eight available LVNC hearts. The genetic yield was 34.2%. Patients with negative genetic testing had better left ventricular ejection fraction (LVEF) or it showed a tendency to improve in follow-up, and a possible trigger factor for LVNC was identified in 1/3 of them. Nonetheless, cerebrovascular accidents occurred in similar proportions in both groups. We conclude that in LVNC there seem to be different ways to achieve the same final phenotype. Genetic testing has a good genetic yield and provides valuable information. LVNC without an underlying genetic cause may have a better prognosis in terms of LVEF evolution. However, anticoagulation to prevent cerebrovascular accident (CVA) should be carefully evaluated in all patients. Larger series with pathologic examination are needed to help better understand this entity.

Genetic contribution of endoplasmic reticulum aminopeptidase 1 polymorphisms to liver fibrosis progression in patients with HCV infection.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses, trimming peptides and loading onto HLA class I molecules. Coding single nucleotide polymorphisms within ERAP1 are associated with autoimmune diseases, viral infections, and cancer development. Our purpose was to analyze the influence of ERAP1 variants on fibrogenesis in hepatitis C virus (HCV)-infected patients. A range of ERAP1 polymorphisms were genotyped in 722 unrelated Caucasian patients diagnosed with chronic HCV from two Spanish cohorts. Patients were classified according to their fibrosis stage. Paraffin-embedded tissue microarrays were constructed to assess ERAP1 expression (HCV = 38; alcoholic = 20) by immunohistochemistry. A statistical algorithm was applied to derive a fibrogenesis prediction model. The ERAP1 variants rs30187/T (K528, pc < 0.001) and rs27044/G (Q730, pc < 0.001) were related with severe fibrosis. These results were validated in the two independent cohorts. Furthermore, patients with the rs30187/T allele had stronger ERAP1 protein expression than those with the rs30187/C (p < 0.05). The statistical model showed that patients with rs30187 C/T and T/T genotypes took 15.58 years (median) to develop advanced fibrosis, but this value was 32.08 years in patients carrying C/C genotype (p < 0.005). ERAP1 variants may influence the clinical course of fibrogenesis in HCV-infected patients. These polymorphisms could be exploited as constitutive new markers of fibrosis evolution. The results highlight the possibility of using modulators of ERAP1 to generate a protective immune response against chronic HCV infection. KEY MESSAGES: What is known Several ERAP1 polymorphisms are associated with autoimmune diseases and cancer. ERAP1 trims peptides to HLA class I presentation. What is new here ERAP1 polymorphisms are associated with fibrogenesis. The ERAP1 polymorphisms genotype could help us in clinical management of patients. Potential translational impact The use of modulators of ERAP1 could generate a protective response depending on SNPs.

Epigenetic downregulation of TET3 reduces genome-wide 5hmC levels and promotes glioblastoma tumorigenesis.

Loss of 5-hydroxymethylcytosine (5hmC) has been associated with mutations of the ten-eleven translocation (TET) enzymes in several types of cancer. However, tumors with wild-type TET genes can also display low 5hmC levels, suggesting that other mechanisms involved in gene regulation might be implicated in the decline of this epigenetic mark. Here we show that DNA hypermethylation and loss of DNA hydroxymethylation, as well as a marked reduction of activating histone marks in the TET3 gene, impair TET3 expression and lead to a genome-wide reduction in 5hmC levels in glioma samples and cancer cell lines. Epigenetic drugs increased expression of TET3 in glioblastoma cells and ectopic overexpression of TET3 impaired in vitro cell growth and markedly reduced tumor formation in immunodeficient mice models. TET3 overexpression partially restored the genome-wide patterns of 5hmC characteristic of control brain samples in glioblastoma cell lines, while elevated TET3 mRNA levels were correlated with better prognosis in glioma samples. Our results suggest that epigenetic repression of TET3 might promote glioblastoma tumorigenesis through the genome-wide alteration of 5hmC.

Epigenetic Deregulation of Protocadherin PCDHGC3 in Pheochromocytomas/Paragangliomas Associated With SDHB Mutations.

CONTEXT: SDHB mutations are found in an increasing number of neoplasms, most notably in paragangliomas and pheochromocytomas (PPGLs). SDHB-PPGLs are slow-growing tumors, but ∼50% of them may develop metastasis. The molecular basis of metastasis in these tumors is a long-standing and unresolved problem. Thus, a better understanding of the biology of metastasis is needed. OBJECTIVE: This study aimed to identify gene methylation changes relevant for metastatic SDHB-PPGLs. DESIGN: We performed genome-wide profiling of DNA methylation in diverse clinical and genetic PPGL subtypes, and validated protocadherin γ-C3 (PCDHGC3) gene promoter methylation in metastatic SDHB-PPGLs. RESULTS: We define an epigenetic landscape specific for metastatic SDHB-PPGLs. DNA methylation levels were found significantly higher in metastatic SDHB-PPGLs than in SDHB-PPGLs without metastases. One such change included long-range de novo methylation of the PCDHA, PCDHB, and PCDHG gene clusters. High levels of PCDHGC3 promoter methylation were validated in primary metastatic SDHB-PPGLs, it was found amplified in the corresponding metastases, and it was significantly correlated with PCDHGC3 reduced expression. Interestingly, this epigenetic alteration could be detected in primary tumors that developed metastasis several years later. We also show that PCDHGC3 down regulation engages metastasis-initiating capabilities by promoting cell proliferation, migration, and invasion. CONCLUSIONS: Our data provide a map of the DNA methylome episignature specific to an SDHB-mutated cancer and establish PCDHGC3 as a putative suppressor gene and a potential biomarker to identify patients with SDHB-mutated cancer at high risk of metastasis who might benefit from future targeted therapies.

New Chondrosarcoma Cell Lines with Preserved Stem Cell Properties to Study the Genomic Drift During In Vitro/In Vivo Growth.

For the cancer genomics era, there is a need for clinically annotated close-to-patient cell lines suitable to investigate altered pathways and serve as high-throughput drug-screening platforms. This is particularly important for drug-resistant tumors like chondrosarcoma which has few models available. Here we established and characterized new cell lines derived from two secondary (CDS06 and CDS11) and one dedifferentiated (CDS-17) chondrosarcomas as well as another line derived from a CDS-17-generated xenograft (T-CDS17). These lines displayed cancer stem cell-related and invasive features and were able to initiate subcutaneous and/or orthotopic animal models. Different mutations in Isocitrate Dehydrogenase-1 (IDH1), Isocitrate Dehydrogenase-2 (IDH2), and Tumor Supressor P53 (TP53) and deletion of Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) were detected both in cell lines and tumor samples. In addition, other mutations in TP53 and the amplification of Mouse Double Minute 2 homolog (MDM2) arose during cell culture in CDS17 cells. Whole exome sequencing analysis of CDS17, T-CDS17, and matched patient samples confirmed that cell lines kept the most relevant mutations of the tumor, uncovered new mutations and revealed structural variants that emerged during in vitro/in vivo growth. Altogether, this work expanded the panel of clinically and genetically-annotated chondrosarcoma lines amenable for in vivo studies and cancer stem cell (CSC) characterization. Moreover, it provided clues of the genetic drift of chondrosarcoma cells during the adaptation to grow conditions.

Laser ablation ICP-MS for simultaneous quantitative imaging of iron and ferroportin in hippocampus of human brain tissues with Alzheimer's disease.

Laser ablation inductively coupled plasma - mass spectrometry (LA-ICP-MS) is proposed for a better understanding of metals and proteins distribution in micrometre structures of human brain tissues. Simultaneous absolute quantitative imaging of Fe and ferroportin (FPN), in 5 µm thick tissue sections of the stratum pyramidale of hippocampus CA1 region, was carried out for Alzheimer disease (AD) patients and healthy controls (HC). For the imaging of FPN by LA-ICP-MS, antibodies were labelled via carbodiimide crosslinking with fluorescent gold nanoclusters (AuNCs) of 2.2 nm diameter, enabling a high amplification (314 gold atoms per NC). Laboratory made gelatin standards containing Fe and Au were used for LA-ICP-MS calibration. Results showed that iron presents an increased concentration in AD donors compared with HC donors, whereas similar concentrations of FPN in AD donors with respect to HC donors were obtained. The average absolute FPN concentrations in selected areas obtained with the proposed AuNCs method were compared with the levels obtained by densitometric analysis with a traditional IHC approach, observing a similar trend in all cases.