High frequency and founder effect of the CYP3A4*20 loss-of-function allele in the Spanish population classifies CYP3A4 as a polymorphic enzyme.

Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4000 individuals representing different populations, and haplotype analysis was performed using CYP3A polymorphisms and microsatellite markers. CYP3A4*20 allele was present in 1.2% of the Spanish population (up to 3.8% in specific regions), and all CYP3A4*20 carriers had a common haplotype. This is compatible with a Spanish founder effect and classifies CYP3A4 as a polymorphic enzyme. This constitutes the first description of a CYP3A4 loss-of-function variant with high frequency in a population. CYP3A4*20 results together with the key role of CYP3A4 in drug metabolism support screening for rare CYP3A4 functional alleles among subjects with adverse drug events in certain populations.This work was supported by projects from the Spanish Ministry of Economy and Competiveness (grant number SAF2012-35779). Government of Extremadura-AEXCID (13/A001), the RIBEF IberoAmerican Network of Pharmacogenetics and SIFF (http: //www.ribef.com). MA-R and VM are predoctoral fellows of 'la Caixa'/ CNIO international PhD programme. LI-P is supported by CIBERER. MC is a predoctoral fellow supported by Severo Ochoa. AAdC is supported by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 259735. MEGN is supported by the European Union (FSE), Gobierno de Extremadura and Consejeria de Empleo, Empresa e Innovacion Grant PD10199.S

Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways.

Pheochromocytoma (PCC) and paraganglioma (PGL) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. Since microRNAs are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of microRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified microRNA signatures specific to, as well as common among, the genetic groups of PCC/PGLs. MicroRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD- and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using qRT-PCR. Up-regulation of miR-210 in VHL- and SDHB-related PCC/PGL, while miR-137 and miR-382 were confirmed as generally up-regulated in PCC/PGL (except in MAX-related tumors). Also, we confirmed over-expression of miR-133b as VHL-specific, miR-488 and miR-885-5p as RET-specific, and miR-183 and miR-96 as SDHB-specific microRNAs.To determine the potential roles microRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX-tumors allowed us to determine that microRNA regulation occurs primarily through mRNA degradation in PCC/PGL, which partially confirmed our miRNA-mRNA integration results

NK cell-triggered CCL5/IFNγ-CXCL9/10 axis underlies the clinical efficacy of neoadjuvant anti-HER2 antibodies in breast cancer

Background: The variability in responses to neoadjuvant treatment with anti-HER2 antibodies prompts to personalized clinical management and the development of innovative treatment strategies. Tumor-infiltrating Natural Killer (TI-NK) cells can predict the efficacy of HER2-targeted antibodies independently from clinicopathological factors in primary HER2-positive breast cancer patients. Understanding the mechanism/s underlying this association would contribute to optimizing patient stratification and provide the rationale for combinatorial approaches with immunotherapy. Methods: We sought to uncover processes enriched in NK cell-infiltrated tumors as compared to NK cell-desert tumors by microarray analysis. Findings were validated in clinical trial-derived transcriptomic data. In vitro and in vivo preclinical models were used for mechanistic studies. Findings were analysed in clinical samples (tumor and serum) from breast cancer patients. Results: NK cell-infiltrated tumors were enriched in CCL5/IFNG-CXCL9/10 transcripts. In multivariate logistic regression analysis, IFNG levels underlie the association between TI-NK cells and pathological complete response to neoadjuvant treatment with trastuzumab. Mechanistically, the production of IFN-ɣ by CD16+ NK cells triggered the secretion of CXCL9/10 from cancer cells. This effect was associated to tumor growth control and the conversion of CD16 into CD16-CD103+ NK cells in humanized in vivo models. In human breast tumors, the CD16 and CD103 markers identified lineage-related NK cell subpopulations capable of producing CCL5 and IFN-ɣ, which correlated with tissue-resident CD8+ T cells. Finally, an early increase in serum CCL5/CXCL9 levels identified patients with NK cell-rich tumors showing good responses to anti-HER2 antibody-based neoadjuvant treatment. Conclusions: This study identifies specialized NK cell subsets as the source of IFN-ɣ influencing the clinical efficacy of anti-HER2 antibodies. It also reveals the potential of serum CCL5/CXCL9 as biomarkers for identifying patients with NK cell-rich tumors and favorable responses to anti-HER2 antibody-based neoadjuvant treatment.AM is supported by ISCiii/FEDER (PI19/00328, PI22/00040 and CIBERONC). The authors are supported by coordinated research projects from Asociación Española contra el Cáncer (GCB15152947MELE). AM and MLB are supported by Generalitat de Catalunya (2017 SGR 888 and 2023 SGR 863). ARe is funded by EC Horizon 2020. Marie Sklodowska Curie-Innovative Training Network (No. 765104; 2018–2021). JA, FR and AR are supported by ISCiii/FEDER (PI18/00006; PI21/00002 and CIBERONC) and by Generalitat de Catalunya (2017 SGR 507; 2021 SGR00776)

Gain-of-function mutations in DNMT3A in patients with paraganglioma

Purpose: 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. Methods: 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. Results: 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. Conclusion: 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