Extracellular Vesicles-A Source of RNA Biomarkers for the Detection of Breast Cancer in Liquid Biopsies.

Over the past decade, extracellular vesicles (EVs) have emerged as a promising source of cancer-derived RNAs for liquid biopsies. However, blood contains a pool of heterogeneous EVs released by a variety of cell types, making the identification of cancer RNA biomarkers challenging. Here, we performed deep sequencing of plasma EV RNA cargo in 32 patients with locally advanced breast cancer (BC) at diagnosis and 7 days after breast surgery and in 30 cancer-free healthy controls (HCs). To identify BC-derived RNA biomarkers, we searched for RNAs that had higher levels in BC EVs at the time of diagnosis compared with HCs and decreased after surgery. Data analysis showed that the fractions of miRNAs, snRNAs, snoRNAs, and tRFs were increased, but the fraction of lncRNAs was decreased in BC EVs as compared to HCs. BC-derived biomarker candidates were identified across various RNA biotypes. Considered individually, they had very high specificity but moderate sensitivity for the detection of BC, whereas a biomarker model composed of eight RNAs: SNORD3H, SNORD1C, SNORA74D, miR-224-5p, piR-32949, lnc-IFT-122-2, lnc-C9orf50-4, and lnc-FAM122C-3 was able to distinguish BC from HC EVs with an AUC of 0.902 (95% CI = 0.872-0.931, p = 3.4 × 10-9) in leave-one-out cross-validation. Furthermore, a number of RNA biomarkers were correlated with the ER and HER2 expression and additional biomarker models were created to predict hormone receptor and HER2 status. Overall, this study demonstrated that the RNA composition of plasma EVs is altered in BC patients and that they contain cancer-derived RNA biomarkers that can be used for BC detection and monitoring using liquid biopsies.

Plasma and urinary extracellular vesicles as a source of RNA biomarkers for prostate cancer in liquid biopsies.

Introduction: Extracellular vesicles (EVs) have emerged as a very attractive source of cancer- derived RNA biomarkers for the early detection, prognosis and monitoring of various cancers, including prostate cancer (PC). However, biofluids contain a mixture of EVs released from a variety of tissues and the fraction of total EVs that are derived from PC tissue is not known. Moreover, the optimal biofluid-plasma or urine-that is more suitable for the detection of EV- enclosed RNA biomarkers is not yet clear. Methodology: In the current study, we performed RNA sequencing analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer. Results and Discussion: The most abundant RNA biotypes in EVs were miRNA, piRNA, tRNA, lncRNA, rRNA and mRNA. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype. The levels of 63 mRNAs, 3 lncRNAs, 2 miRNAs and 1 piRNA were significantly increased in the tumors and decreased in the post-operation urinary EVs, thus suggesting that these RNAs mainly originate from PC tissue. No such RNA biomarkers were identified in plasma EVs. This suggests that the fraction of PC-derived EVs in urine is larger than in plasma and allows the detection and tracking of PC-derived RNAs.

Comprehensive characterization of RNA cargo of extracellular vesicles in breast cancer patients undergoing neoadjuvant chemotherapy.

Extracellular vesicles (EVs) are g7aining increased attention as carriers of cancer-derived molecules for liquid biopsies. Here, we studied the dynamics of EV levels in the plasma of breast cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC) and explored the relevance of their RNA cargo for the prediction of patients' response to the therapy. EVs were isolated from serial blood samples collected at the time of diagnosis, at the end of NAC, and 7 days, 6, and 12 months after the surgery from 32 patients with locally advanced BC, and 30 cancer-free healthy controls (HCs) and quantified by nanoparticle tracking analysis. The pre-treatment levels of EVs in BC patients were higher than in HCs, significantly increased during the NAC and surgery, and decreased to the levels found in HCs 6 months after surgery, thus showing that a substantial fraction of plasma EVs in BC patients are produced due to the disease processes and treatment. RNA sequencing analysis revealed that the changes in the EV levels were associated with the alterations in the proportions of various RNA biotypes in EVs. To search for RNA biomarkers that predict response to the NAC, patients were dichotomized as responders and non-responders based on Miller-Payne grades and differential expression analyses were carried out between responders and non-responders, and HCs. This resulted in the identification of 6 miRNAs, 4 lncRNAs, and 1 snoRNA that had significantly higher levels in EVs from non-responders than responders at the time of diagnosis and throughout the NAC, and significantly lower levels in HCs, thus representing biomarkers for the prediction of response to NAC at the time of diagnosis. In addition, we found 14 RNAs representing piRNA, miRNA, lncRNA, snoRNA, and snRNA biotypes that were induced by NAC in non-responders and 2 snoRNAs and 1 piRNA that were induced by NAC in patients with early disease progression, thus warranting further functional studies on their role in chemoresistance and metastasis.

A novel EDA variant causing X-linked hypohidrotic ectodermal dysplasia: Case report.

Hereditary ectodermal dysplasias are a complex group of inherited disorders characterised by abnormalities in two or more ectodermal derivatives (skin, nails, sweat glands, etc.). There are two main types of these disorders - hidrotic and hypohidrotic/anhidrotic ectodermal dysplasias. Hypohidrotic ectodermal dysplasia (HED) or Christ-Siemens-Touraine syndrome (OMIM: 305100) occurs in 1 out of 5000-10,000 births [19] and has an X-linked recessive inheritance pattern (X-linked hypohydrotic ectodermal dysplasia - XLHED) [2]. The main cause of XLHED is a broad range of pathogenic variants in the EDA gene (HGNC:3157, Xq12-13) which encodes the transmembrane protein ectodysplasin-A [4]. We report here the case of a patient with a novel inherited allelic variant in the EDA gene - NM_001399.5:c.337C>T (p.Gln113*) - in the heterozygous state. Targeted family member screening was conducted and other carriers of this EDA gene pathogenic variant were identified and phenotypically characterised. The patient subsequently underwent in vitro fertilisation with preimplantation genetic testing for monogenic diseases (PGT-M).

Case report: recurrent pituitary adenoma has increased load of somatic variants.

BACKGROUND: Pituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. In this study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in the same patient to explain the causality mechanisms of PA recurrence. CASE PRESENTATION: The patient was a 69-year-old female with non-functional pituitary macroadenoma with extension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August 2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knosp grade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In both tumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germline DNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling were performed followed by variant filtering and manual review with IGV software. We observed an increased load of missense variants in the recurrent PA tumour when compared to the original tumour. The number of detected variants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally, targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encoded proteins. CONCLUSIONS: In this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression. The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused by clonal expansion of the leftover tumour tissue.

Significantly altered peripheral blood cell DNA methylation profile as a result of immediate effect of metformin use in healthy individuals.

BACKGROUND: Metformin is a widely prescribed antihyperglycemic agent that has been also associated with multiple therapeutic effects in various diseases, including several types of malignancies. There is growing evidence regarding the contribution of the epigenetic mechanisms in reaching metformin's therapeutic goals; however, the effect of metformin on human cells in vivo is not comprehensively studied. The aim of our study was to examine metformin-induced alterations of DNA methylation profiles in white blood cells of healthy volunteers, employing a longitudinal study design. RESULTS: Twelve healthy metformin-naïve individuals where enrolled in the study. Genome-wide DNA methylation pattern was estimated at baseline, 10 h and 7 days after the start of metformin administration. The whole-genome DNA methylation analysis in total revealed 125 differentially methylated CpGs, of which 11 CpGs and their associated genes with the most consistent changes in the DNA methylation profile were selected: POFUT2, CAMKK1, EML3, KIAA1614, UPF1, MUC4, LOC727982, SIX3, ADAM8, SNORD12B, VPS8, and several differentially methylated regions as novel potential epigenetic targets of metformin. The main functions of the majority of top-ranked differentially methylated loci and their representative cell signaling pathways were linked to the well-known metformin therapy targets: regulatory processes of energy homeostasis, inflammatory responses, tumorigenesis, and neurodegenerative diseases. CONCLUSIONS: Here we demonstrate for the first time the immediate effect of short-term metformin administration at therapeutic doses on epigenetic regulation in human white blood cells. These findings suggest the DNA methylation process as one of the mechanisms involved in the action of metformin, thereby revealing novel targets and directions of the molecular mechanisms underlying the various beneficial effects of metformin. TRIAL REGISTRATION: EU Clinical Trials Register, 2016-001092-74. Registered 23 March 2017, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001092-74/LV .

Next-generation-sequencing-based identification of familial hypercholesterolemia-related mutations in subjects with increased LDL-C levels in a latvian population.

BACKGROUND: Familial hypercholesterolemia (FH) is one of the commonest monogenic disorders, predominantly inherited as an autosomal dominant trait. When untreated, it results in early coronary heart disease. The vast majority of FH remains undiagnosed in Latvia. The identification and early treatment of affected individuals remain a challenge worldwide. Most cases of FH are caused by mutations in one of four genes, APOB, LDLR, PCSK9, or LDLRAP1. The spectrum of disease-causing variants is very diverse and the variation detection panels usually used in its diagnosis cover only a minority of the disease-causing gene variants. However, DNA-based tests may provide an FH diagnosis for FH patients with no physical symptoms and with no known family history of the disease. Here, we evaluate the use of targeted next-generation sequencing (NGS) to identify cases of FH in a cohort of patients with coronary artery disease (CAD) and individuals with abnormal low-density lipoprotein-cholesterol (LDL-C) levels. METHODS: We used targeted amplification of the coding regions of LDLR, APOB, PCSK9, and LDLRAP1, followed by NGS, in 42 CAD patients (LDL-C, 4.1-7.2 mmol/L) and 50 individuals from a population-based cohort (LDL-C, 5.1-9.7 mmol/L). RESULTS: In total, 22 synonymous and 31 nonsynonymous variants, eight variants in close proximity (10 bp) to intron-exon boundaries, and 50 other variants were found. We identified four pathogenic mutations (p.(Arg3527Gln) in APOB, and p.(Gly20Arg), p.(Arg350*), and c.1706-10G > A in LDLR) in seven patients (7.6 %). Three possible pathogenic variants were also found in four patients. CONCLUSION: NGS-based methods can be used to detect FH in high-risk individuals when they do not meet the defined clinical criteria.