Rationale : 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 in vitro models, and define specific therapeutic options according to tumor genomic features. Methods : 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 in vitro . Results : 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, P =4.67·10 -18 ), and was found associated in vitro 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 in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions : 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.
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.
Exome sequencing is utilized in routine clinical genetic diagnosis. The technical robustness of repurposing large-scale next-generation sequencing data for pharmacogenetics has been demonstrated, supporting the implementation of preemptive pharmacogenetic strategies based on adding clinical pharmacogenetic interpretation to exomes. However, a comprehensive study analyzing all actionable pharmacogenetic alleles contained in international guidelines and applied to diagnostic exome data has not been performed. Here, we carried out a systematic analysis based on 5001 Spanish or Latin American individuals with diagnostic exome data, either Whole Exome Sequencing (80%), or the so-called Clinical Exome Sequencing (20%) (60 Mb and 17 Mb, respectively), to provide with global and gene-specific clinical pharmacogenetic utility data. 788 pharmacogenetic alleles, distributed through 19 genes included in Clinical Pharmacogenetics Implementation Consortium guidelines were analyzed. We established that Whole Exome and Clinical Exome Sequencing performed similarly, and 280 alleles in 11 genes (CACNA1S, CYP2B6, CYP2C9, CYP4F2, DPYD, G6PD, NUDT15, RYR1, SLCO1B1, TPMT, and UGT1A1) could be used to inform of pharmacogenetic phenotypes that change drug prescription. Each individual carried in average 2.2 alleles and overall 95% (n = 4646) of the cohort could be informed of at least one actionable pharmacogenetic phenotype. Differences in variant allele frequency were observed among the populations studied and the corresponding gnomAD population for 7.9% of the variants. In addition, in the 11 selected genes we uncovered 197 novel variants, among which 27 were loss-of-function. In conclusion, we provide with the landscape of actionable pharmacogenetic information contained in diagnostic exomes, that can be used preemptively in the clinics.
The mammalian target of rapamycin (mTOR) pathway inhibitors are key drugs for the treatment of many tumor types, however, there are no predictive biomarkers in clinical use. Here, we performed a molecular and immunohistochemical characterization of key mTOR pathway components in a series of 105 renal cell carcinoma patients treated with rapalogs, aimed at identifying markers of treatment response. Mutational analysis in MTOR, TSC1 and TSC2 was performed through targeted next‐generation sequencing (NGS), and immunohistochemistry (IHC) was performed for PTEN, pAKT, pS6K1, pS6 and p21. Among patients with NGS data, 11 of 87 (13%) had mTOR pathway mutations (8 in MTOR, 1 in TSC1 and 2 in TSC2). When comparing the molecular data to the response of the patients, we found that partial response was more frequent in cases with mTOR pathway mutations than in those without mutations (odds ratio [OR] = 0.08, 95% confidence interval [CI] = 0.008–0.79, p = 0.030 univariate; p = 0.038 multivariable). Regarding IHC, negative PTEN staining was detected in 58% of the tumors, and it was more frequent in rapalog responder patients (OR = 0.24, 95% CI = 0.065–0.86, p = 0.029 univariate; p = 0.029 multivariable). Mutations and PTEN IHC were not mutually exclusive events and its combination improved response prediction (OR = 0.16, 95% CI = 0.04–0.62, p = 0.008 univariate; p = 0.013 multivariable). The staining of other proteins did not show and association with response and no association with PFS was observed in unselected patients. In conclusion, our findings suggest that mTOR pathway mutations, negative PTEN IHC and their combination are potential markers of rapalog response.
Medical writing and statistical analyses were performed by Pau Doñate Ph.D. and Jordi Curto M.Sc., respectively, both from MFAR Clinical Research (Barcelona, Spain).GETNE-1408 trial was sponsored by Grupo Español de Tumores Neuroendocrinos y Endocrinos (GETNE). Threshold/MTEM provided evofosfamide and awarded a grant to GETNE to pay the costs of the study. Pfizer provided sunitinib.
Context The identification of markers able to determine Medullary Thyroid Cancer (MTC) patients at high-risk of disease progression is critical to improve their clinical management and outcome. Previous studies have suggested that expression of the stem-cell marker CD133 is associated with MTC aggressiveness. Objective To evaluate CD133 impact on disease progression in MTC and explore the regulatory mechanisms leading to the upregulation of this protein in aggressive tumors. Patients We compiled a series of 74 MTCs with associated clinical data and characterized them for mutations in RET and RAS proto-oncogenes, presumed to be related with disease clinical behavior. Results We found that CD133 immunohistochemical expression was associated with adverse clinicopathological features and predicted a reduction in time to disease progression even when only RET-mutated cases were considered in the analysis (log-rank test p<0.003). Univariate analysis for progression-free survival revealed CD133 expression and presence of tumor emboli in peritumoral blood vessels as the most significant prognostic covariates among others such as age, gender and prognostic stage. Multivariate analysis identified both variables as independent factors of poor prognosis (hazard ratio=16.6 and 2; p=0.001 and 0.010, respectively). Finally, we defined hsa-miR-30a-5p, a miRNA downregulated in aggressive MTCs, as a CD133 expression regulator. Ectopic expression of hsa-miR-30a-5p in MZ-CRC-1 (RETM918T) cells significantly reduced CD133 mRNA expression. Conclusions Our results suggest that CD133 expression may be a useful tool to identify MTC patients with poor prognosis, who may benefit from a more extensive primary surgical management and follow-up.
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.
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