The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

The pathological implication of amyloid precursor protein (APP) in Alzheimer’s disease has been widely documented due to its involvement in the generation of amyloid-β peptide. However, the physiological functions of APP are still poorly understood. APP is considered a multimodal protein due to its role in a wide variety of processes, both in the embryo and in the adult brain. Specifically, APP seems to play a key role in the proliferation, differentiation and maturation of neural stem cells. In addition, APP can be processed through two canonical processing pathways, generating different functionally active fragments: soluble APP-α, soluble APP-β, amyloid-β peptide and the APP intracellular C-terminal domain. These fragments also appear to modulate various functions in neural stem cells, including the processes of proliferation, neurogenesis, gliogenesis or cell death. However, the molecular mechanisms involved in these effects are still unclear. In this review, we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells, as well as the possible signaling pathways that could be implicated in these effects. The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer’s disease is essential to advance the understanding of the pathogenesis of Alzheimer’s disease, and in the search for potential therapeutic targets.

Background: Approximately 15% of adult GIST patients harbor tumors that are wild-type for KIT and PDGFRα genes (KP-wtGIST). These tumors usually have SDH deficiencies, exhibit a more indolent behavior and are resistant to imatinib. Underlying oncogenic mechanisms in KP-wtGIST include overexpression of HIF1α high IGFR signaling through the MAPK pathway or BRAF activating mutation, among others. As regorafenib inhibits these signaling pathways, it was hypothesized that it could be more active as upfront therapy in advanced KP-wtGIST. Methods: Adult patients with advanced KP-wtGIST after central confirmation by NGS, naïve of systemic treatment for advanced disease, were included in this international phase II trial. Eligible patients received regorafenib 160 mg per day for 21 days every 28 days. The primary endpoint was disease control rate (DCR), according to RECIST 1.1 at 12 weeks by central radiological assessment. Results: From May 2016 to October 2020, 30 patients were identified as KP-wtGIST by Sanger sequencing and 16 were confirmed by central molecular screening with NGS. Finally, 15 were enrolled and received regorafenib. The study was prematurely closed due to the low accrual worsened by COVID outbreak. The DCR at 12 weeks was 86.7% by central assessment. A subset of 60% experienced some tumor shrinkage, with partial responses and stabilization observed in 13% and 87% respectively, by central assessment. SDH-deficient GIST showed better clinical outcome than other KP-wtGIST. Conclusions: Regorafenib activity in KP-wtGIST compares favorably with other tyrosine kinase inhibitors, especially in the SDH-deficient GIST subset and it should be taken into consideration as upfront therapy of advanced KP-wtGIST. Trial registration: ClinicalTrials.gov Identifier: NCT02638766.

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients.

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.

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 (r_customPanel = 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.