In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

The molecular genetics of inherited phaeochromocytoma have received considerable attention, but the somatic genetic and epigenetic events that characterise tumourigenesis in sporadic phaeochromocytomas are less well defined. Previously, we found considerable overlap between patterns of promoter region tumour suppressor gene (TSG) hypermethylation in two neural crest tumours, neuroblastoma and phaeochromocytoma. In order to identify candidate biomarkers and epigenetically inactivated TSGs in phaeochromocytoma and neuroblastoma, we characterised changes in gene expression in three neuroblastoma cell lines after treatment with the demethylating agent 5-azacytidine. Promoter region methylation status was then determined for 28 genes that demonstrated increased expression after demethylation. Three genes HSP47, homeobox A9 (HOXA9) and opioid binding protein (OPCML) were methylated in >10% of phaeochromocytomas (52, 17 and 12% respectively). Two of the genes, epithelial membrane protein 3 (EMP3) and HSP47, demonstrated significantly more frequent methylation in neuroblastoma than phaeochromocytoma. These findings extend epigenotype of phaeochromocytoma and identify candidate genes implicated in sporadic phaeochromocytoma tumourigenesis.

Cyclooxygenase 2 (COX-2) is a key enzyme of the tumorigenesis-inflammation interface and can be induced by hypoxia. A pseudohypoxic transcriptional signature characterizes pheochromocytomas and paragangliomas (PPGLs) of the cluster I, mainly represented by tumors with mutations in von Hippel-Lindau (VHL), endothelial PAS domain-containing protein 1 (EPAS1), or succinate dehydrogenase (SDH) subunit genes. The aim of this study was to investigate a possible association between underlying tumor driver mutations and COX-2 in PPGLs. COX-2 gene expression and immunoreactivity were examined in clinical specimens with documented mutations, as well as in spheroids and allografts derived from mouse pheochromocytoma (MPC) cells. COX-2 in vivo imaging was performed in allograft mice. We observed significantly higher COX-2 expression in cluster I, especially in VHL-mutant PPGLs, however, no specific association between COX-2 mRNA levels and a hypoxia-related transcriptional signature was found. COX-2 immunoreactivity was present in about 60% of clinical specimens as well as in MPC spheroids and allografts. A selective COX-2 tracer specifically accumulated in MPC allografts. This study demonstrates that, although pseudohypoxia is not the major determinant for high COX-2 levels in PPGLs, COX-2 is a relevant molecular target. This potentially allows for employing selective COX-2 inhibitors as targeted chemotherapeutic agents and radiosensitizers. Moreover, available models are suitable for preclinical testing of these treatments.

Over the past few years, next generation technologies have been applied to unravel the genetics of rare inherited diseases, facilitating the discovery of new susceptibility genes. We recently found germline DNMT3A gain-of-function variants in two patients with head and neck paragangliomas causing a characteristic hypermethylated DNA profile. Here, whole-exome sequencing identifies a novel germline DNMT3A variant (p.Gly332Arg) in a patient with bilateral carotid paragangliomas, papillary thyroid carcinoma and idiopathic intellectual disability. The variant, located in the Pro-Trp-Trp-Pro (PWWP) domain of the protein involved in chromatin targeting, affects a residue mutated in papillary thyroid tumors and located between the two residues found mutated in microcephalic dwarfism patients. Structural modelling of the variant in the DNMT3A PWWP domain predicts that the interaction with H3K36me3 will be altered. An increased methylation of DNMT3A target genes, compatible with a gain-of-function effect of the alteration, was observed in saliva DNA from the proband and in one independent acute myeloid leukemia sample carrying the same p.Gly332Arg variant. Although further studies are needed to support a causal role of DNMT3A variants in paraganglioma, the description of a new DNMT3A alteration in a patient with multiple clinical features suggests a heterogeneous phenotypic spectrum related to DNMT3A germline variants.

Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) is a very rare hereditary disorder characterized by cutaneous leiomyomas (CLMs), uterine leiomyomas (ULMs), renal cysts (RCys) and renal cell cancers (RCCs). We aimed to describe the genetics, clinical features and potential genotype-phenotype associations in the largest cohort of fumarate hydratase enzyme mutation carriers known from Spain using a multicentre, retrospective study of individuals with a genetic or clinical diagnosis of HLRCC. We collected clinical information from medical records, analysed genetic variants and looked for genotype-phenotype associations. Analyses were performed using R 3.6.0. software. We included 197 individuals: 74 index cases and 123 relatives. CLMs were diagnosed in 65% of patients, ULMs in 90% of women, RCys in 37% and RCC in 10.9%. Twenty-seven different pathogenic variants were detected, 12 (44%) of them not reported previously. Patients with missense pathogenic variants showed higher frequencies of CLMs, ULMs and RCys, than those with loss-of-function variants (p = 0.0380, p = 0.0015 and p = 0.024, respectively). This is the first report of patients with HLRCC from Spain. The frequency of RCCs was lower than those reported in the previously published series. Individuals with missense pathogenic variants had higher frequencies of CLMs, ULMs and RCys.

Metastatic breast cancer (MBC) progressing after endocrine therapy frequently activates PI3K/AKT/mTOR pathway. The BOLERO-2 trial showed that everolimus-exemestane achieves increased progression free survival (PFS) compared with exemestane. However, there is great inter-patient variability in toxicity and response to exemestane-everolimus treatment. The objective of this study was to perform an exploratory study analyzing the implication of single nucleotide polymorphisms (SNPs) on outcomes from this treatment through a pharmacogenetic analysis.

Specific genetic variants in the mitochondrially encoded 12S ribosomal RNA gene (MT-RNR1) cause aminoglycoside-induced irreversible hearing loss. Mitochondrial DNA is usually not included in targeted sequencing experiments; however, off-target data may deliver this information. Here, we extract MT-RNR1 genetic variation, including the most relevant ototoxicity variant m.1555A>G, using the off-target reads of 473 research samples, sequenced through a capture-based, custom-targeted panel and whole exome sequencing (WES), and of 1245 diagnostic samples with clinical WES. Sanger sequencing and fluorescence-based genotyping were used for genotype validation. There was a correlation between off-target reads and mitochondrial coverage (rcustomPanel = 0.39, p = 2 × 10-13 and rWES = 0.67, p = 7 × 10-21). The median read depth of MT-RNR1 m.1555 was similar to the average mitochondrial genome coverage, with saliva and blood samples giving comparable results. The genotypes from 415 samples, including three m.1555G carriers, were concordant with fluorescence-based genotyping data. In clinical WES, median MT-RNR1 coverage was 56×, with 90% of samples having ≥20 reads at m.1555 position, and one m.1494T and three m.1555G carriers were identified with no evidence for heteroplasmy. Altogether, this study shows that obtaining MT-RNR1 genotypes through off-target reads is an efficient strategy that can impulse preemptive pharmacogenetic screening of this mitochondrial gene.

Pheochromocytomas/paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors.

Combination therapies with gut hormone analogs represent promising treatment strategies for obesity. This pilot study investigates the therapeutic potential of modulators of the glucagon-like peptide 1 (GLP-1) and neuropeptide Y (NPY) system using GLP-1 receptor agonists (semaglutide) and antagonists (exendin 9-39), as well as non-selective and NPY-Y2-receptor selective peptide tyrosine tyrosine (PYY) analogs (PYY3-36/NNC0165-0020 and NNC0165-1273) and an NPY-Y2 receptor antagonist (JNJ31020028).

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors with a strong hereditary background and a large genetic heterogeneity. Identification of the underlying genetic cause is crucial for the management of patients and their families as it aids differentiation between hereditary and sporadic cases. To improve diagnostics and clinical management we tailored an enrichment based comprehensive multi-gene next generation sequencing panel applicable to both analyses of tumor tissue and blood samples. We applied this panel to tumor samples and compared its performance to our current routine diagnostic approach. Routine diagnostic sequencing of 11 PPGL susceptibility genes was applied to blood samples of 65 unselected PPGL patients at a single center in Dresden, Germany. Predisposing germline mutations were identified in 19 (29.2%) patients. Analyses of 28 PPGL tumor tissues using the dedicated PPGL panel revealed pathogenic or likely pathogenic variants in known PPGL susceptibility genes in 21 (75%) cases, including mutations in IDH2, ATRX and HRAS. These mutations suggest sporadic tumor development. Our results imply a diagnostic benefit from extended molecular tumor testing of PPGLs and consequent improvement of patient management. The approach is promising for determination of prognostic biomarkers that support therapeutic decision-making.

Papillary Thyroid Cancer (PTC) is a heterogeneous and complex disease; susceptibility to PTC is influenced by the joint effects of multiple common, low-penetrance genes, although relatively few have been identified to date. Here we applied a rigorous combined approach to assess both the individual and epistatic contributions of genetic factors to PTC susceptibility, based on one of the largest series of thyroid cancer cases described to date. In addition to identifying the involvement of TSHR variation in classic PTC, our pioneer study of epistasis revealed a significant interaction between variants in STK17B and PAX8. The interaction was detected by MD-MBR (p = 0.00010) and confirmed by other methods, and then replicated in a second independent series of patients (MD-MBR p = 0.017). Furthermore, we demonstrated an inverse correlation between expression of PAX8 and STK17B in a set of cell lines derived from human thyroid carcinomas. Overall, our work sheds additional light on the genetic basis of thyroid cancer susceptibility, and suggests a new direction for the exploration of the inherited genetic contribution to disease using association studies.

Somatostatin receptor-targeting endoradiotherapy offers potential for treating metastatic pheochromocytomas and paragangliomas, an approach likely to benefit from combination radiosensitization therapy. To provide reliable preclinical in vivo models of metastatic disease, this study characterized the metastatic spread of luciferase-expressing mouse pheochromocytoma (MPC) cells in mouse strains with different immunologic conditions. Bioluminescence imaging showed that, in contrast to subcutaneous non-metastatic engraftment of luciferase-expressing MPC cells in NMRI-nude mice, intravenous cell injection provided only suboptimal metastatic spread in both NMRI-nude mice and hairless SCID (SHO) mice. Treatment of NMRI-nude mice with anti-Asialo GM1 serum enhanced metastatic spread due to substantial depletion of natural killer (NK) cells. However, reproducible metastatic spread was only observed in NK cell-defective SCID/beige mice and in hairless immunocompetent SKH1 mice bearing disseminated or liver metastases, respectively. Liquid chromatography tandem mass spectrometry of urine samples showed that subcutaneous and metastasized tumor models exhibit comparable renal monoamine excretion profiles characterized by increasing urinary dopamine, 3-methoxytyramine, norepinephrine and normetanephrine. Metastases-related epinephrine and metanephrine were only detectable in SCID/beige mice. Positron emission tomography and immunohistochemistry revealed that all metastases maintained somatostatin receptor-specific radiotracer uptake and immunoreactivity, respectively. In conclusion, we demonstrate that intravenous injection of luciferase-expressing MPC cells into SCID/beige and SKH1 mice provides reproducible and clinically relevant spread of catecholamine-producing and somatostatin receptor-positive metastases. These standardized preclinical models allow for precise monitoring of disease progression and should facilitate further investigations on theranostic approaches against metastatic pheochromocytomas and paragangliomas.

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings.

Pheochromocytomas and extra-adrenal paragangliomas (PHEO/PGLs) are rare catecholamine-producing chromaffin cell tumors. For metastatic disease, no effective therapy is available. Overexpression of somatostatin type 2 receptors (SSTR2) in PHEO/PGLs promotes interest in applying therapies using somatostatin analogs linked to radionuclides and/or cytotoxic compounds, such as [(177)Lu]Lu-DOTA-(Tyr(3))octreotate (DOTATATE) and AN-238. Systematic evaluation of such therapies for the treatment of PHEO/PGLs requires sophisticated animal models. In this study, the mouse pheochromocytoma (MPC)-mCherry allograft model showed high tumor densities of murine SSTR2 (mSSTR2) and high tumor uptake of [(64)Cu]Cu-DOTATATE. Using tumor sections, we assessed mSSTR2-specific binding of DOTATATE, AN-238, and somatostatin-14. Therapeutic studies showed substantial reduction of tumor growth and tumor-related renal monoamine excretion in tumor-bearing mice after treatment with [(177)Lu]Lu-DOTATATE compared to AN-238 and doxorubicin. Analyses did not show agonist-dependent receptor downregulation after single mSSTR2-targeting therapies. This study demonstrates that the MPC-mCherry model is a uniquely powerful tool for the preclinical evaluation of SSTR2-targeting theranostic applications in vivo. Our findings highlight the therapeutic potential of somatostatin analogs, especially of [(177)Lu]Lu-DOTATATE, for the treatment of metastatic PHEO/PGLs. Repeated treatment cycles, fractionated combinations of SSTR2-targeting radionuclide and cytotoxic therapies, and other adjuvant compounds addressing additional mechanisms may further enhance therapeutic outcome.

Using an animal model for the study of murine thymic lymphomas, frequent losses of heterozygosity (LOHs) on six chromosomal regions were reported. To determine the existence of LOH loci in human lymphomas, we screened 43 non-Hodgkin's lymphomas by using polymorphisms mapped in each of the orthologous human region. All LOH regions (1p32-p36, 9p21, 9p22-p23, 9q22-q34 and 10q23-q24) were observed in B-cell lymphomas at different frequencies. In addition, analysis of the tumor suppressor genes, p16(INK4a), p73 and PTEN located in 9p21, 1p36 and 10q23, respectively, revealed the participation of p16(INK4a) and p73 but not of PTEN. Importantly, two subregions of LOH were observed in 9q22-q34, one of them not previously described. Our results support the usefulness of murine models to study human lymphoid neoplasias and reveal novel regions on 9q22-q34 candidate for containing tumor suppressor genes involved in human lymphomas.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are neuroendocrine tumors that are mostly benign. Metastatic disease does occur in about 10% of cases of PCC and up to 25% of PGL, and for these patients no effective therapies are available. Patients with mutations in the succinate dehydrogenase subunit B (SDHB) gene tend to have metastatic disease. We hypothesized that a down-regulation in the active succinate dehydrogenase B subunit should result in notable changes in cellular metabolic profile and could present a vulnerability point for successful pharmacological targeting.

Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer.

The limited knowledge about the PTEN hamartoma tumor syndrome (PHTS) makes its diagnosis a challenging task. We aimed to define the clinical and genetic characteristics of this syndrome in the Spanish population and to identify new genes potentially associated with the disease.

Homoeostasis of the autonomic nervous system (ANS) contributes to cognitive functional integrity in learners and can be greatly influenced by emotions and stress. While moderate stress can enhance learning and memory processes, long-term stress compromises learning performance in a face-to-face classroom environment. Integrative online learning and communication tools were shown to be beneficial for visualization and comprehension but their effects on the ANS are poorly understood. We aim to assess the effects of video conference-supported live lectures compared to on-site classroom teaching on autonomic functions and their association with learning performance.