Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors that show the highest heritability of all human neoplasms and represent a paradoxical example of genetic heterogeneity. ...Amongst the elevated number of genes involved in the hereditary predisposition to the disease (at least nineteen) there are eleven tricarboxylic acid (TCA) cycle-related genes, some of which are also involved in the development of congenital recessive neurological disorders and other cancers such as cutaneous and uterine leiomyomas, gastrointestinal tumors and renal cancer. Somatic or germline mutation of genes encoding enzymes catalyzing pivotal steps of the TCA cycle not only disrupts cellular respiration, but also causes severe alterations in mitochondrial metabolite pools. These latter alterations lead to aberrant accumulation of "oncometabolites" that, in the end, may lead to deregulation of the metabolic adaptation of cells to hypoxia, inhibition of the DNA repair processes and overall pathological changes in gene expression. In this review, we will address the TCA cycle mutations leading to the development of PPGL, and we will discuss the relevance of these mutations for the transformation of neural crest-derived cells and potential therapeutic approaches based on the emerging knowledge of underlying molecular alterations.
Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, ...which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.
We used downregulation and methylation of
as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.
One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in
A variant in
, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in
in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline
mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.
This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol.
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It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed ...miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow‐up of 96 months. MiRNome profiles correlated with tumor‐specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa‐miR‐139‐5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease‐free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa‐miR‐139‐5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa‐miR‐139‐5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa‐miR‐139‐5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa‐miR‐139‐5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.
What's new?
Recurrent or metastatic thyroid cancer generally leads to worse outcomes and higher mortality. To search for a prognostic marker in such cases, these authors sequenced the microRNA expression profile, or miRNome, of 46 thyroid samples. They compared differential expression between patients with recurrent/metastatic disease and those who remained cancer free over the 8‐year follow‐up. One miRNA, hsa‐miR‐139‐5p, was associated with recurrent disease independent of genetic background. Expression of hsa‐miR‐139‐5p, they found, influences expression of an alternative splicing factor, HNRNPF, which in turn controls the transcript balance of genes involved in key cancer‐related pathways, a novel mechanism associated with tumor virulence.
The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the ...known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors.
Whole-exome sequencing was applied to the germlines of a parent–proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed.
We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma.
Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.
Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by ...driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.
MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study ...aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype.
Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach.
Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL.
This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.
Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, ...genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.
Background
Peripheral neuropathy is the dose‐limiting toxicity of many oncology drugs, including paclitaxel. There is large interindividual variability in the neuropathy, and several risk factors ...have been proposed; however, many have not been replicated. Here we present a comprehensive study aimed at identifying treatment and physiopathology‐related paclitaxel‐induced neuropathy risk factors in a large cohort of well‐characterized patients.
Patients and Methods
Analyses included 503 patients with breast or ovarian cancer who received paclitaxel treatment. Paclitaxel dose modifications caused by the neuropathy were extracted from medical records and patients self‐reported neuropathy symptoms were collected. Multivariate logistic regression analyses were performed to identify concomitant medications and comorbidities associated with paclitaxel‐induced neuropathy.
Results
Older patients had higher neuropathy: for each increase of 1 year of age, the risk of dose modifications and grade 3 neuropathy increased 4% and 5%, respectively. Cardiovascular drugs increased the risk of paclitaxel dose reductions (odds ratio OR, 2.51; p = .006), with a stronger association for beta‐adrenergic antagonists. The total number of concomitant medications also showed an association with dose modifications (OR, 1.25; p = .012 for each concomitant drug increase). A dose modification predictive model that included the new identified factors gave an area under the curve of 0.74 (p = 1.07 × 10−10). Preexisting nerve compression syndromes seemed to increase neuropathy risk.
Conclusion
Baseline characteristics of the patients, including age and concomitant medications, could be used to identify individuals at high risk of neuropathy, personalizing chemotherapy treatment and reducing the risk of severe neuropathy.
Implications for Practice
Peripheral neuropathy is a common adverse effect of many cancer drugs, including chemotherapeutics, targeted therapies, and immune checkpoint inhibitors. About 40% of survivors of cancer have functional deficits caused by this toxicity, some of them irreversible. Currently, there are no effective treatments to prevent or treat this neuropathy. This study, performed in a large cohort of well‐characterized patients homogenously treated with paclitaxel, identified concomitant medications, comorbidities, and demographic factors associated with peripheral neuropathy. These factors could serve to identify patients at high risk of severe neuropathy for whom alternative non‐neurotoxic alternatives may be considered.
Peripheral neuropathy is a common adverse effect of many cancer drugs, including chemotherapeutics, targeted therapies, and immune checkpoint inhibitors. This article reports results of a study designed to identify treatment and physiopathology‐related paclitaxel‐inducd neuropathy risk factors in a cohort of ore than 500 patients.
: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients ...who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through
models, and define specific therapeutic options according to tumor genomic features.
: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized
.
: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804,
=4.67·10
), and was found associated
with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated
a
repression and an enhanced rapamycin sensitivity upon miR-21-3p expression.
: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.
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