A critical approach for successful use of circulating microRNAs as biomarkers in cardiovascular diseases: the case of hypertrophic cardiomyopathy.

MicroRNAs (miRNAs) are small noncoding RNA molecules that act as major regulators of gene expression at the post-transcriptional level. As the potential applications of miRNAs in the diagnosis and treatment of human diseases have become more evident, many studies of hypertrophic cardiomyopathy (HCM) have focused on the systemic identification and quantification of miRNAs in biofluids and myocardial tissues. HCM is a hereditary cardiomyopathy caused by mutations in genes encoding proteins of the sarcomere. Despite overall improvements in survival, progression to heart failure, stroke, and sudden cardiac death remain prominent features of living with HCM. Several miRNAs have been shown to be promising biomarkers of HCM; however, there are many challenges to ensuring the validity, consistency, and reproducibility of these biomarkers for clinical use. In particular, miRNA testing may be limited by pre-analytical and analytical caveats, making our interpretation of results challenging. Such factors that may affect miRNA testing include sample type selection, hemolysis, platelet activation, and renal dysfunction. Therefore, researchers should be careful when developing appropriate standards for the design of miRNA profiling studies in order to ensure that all results provided are both accurate and reliable. In this review, we discuss the application of miRNAs as biomarkers for HCM.

Dysregulation of Plasma miR-146a and miR-155 Expression Profile in Mycosis Fungoides Is Associated with rs2910164 and rs767649 Polymorphisms.

Diagnosis of Mycosis Fungoides (MF) may be challenging, due to its polymorphic nature. The use of miRNAs as biomarkers to assist in diagnosis has been investigated, mainly in skin lesion biopsies. The purpose of this study is to evaluate the plasma levels of miR-146a and miR-155 in MF patients and to investigate their association with SNPs of their genes. Plasma miRNAs were quantified by RT-qPCR. Genomic DNA was used for SNPs' genotyping by Sanger sequencing. Plasma levels of miR-146a and miR-155 were significantly higher in patients vs. controls, in early MF patients vs. controls, and in advanced vs. early MF patients. Both miRNAs' levels were significantly higher in stage IIB vs. early-stage patients. miR-155 plasma levels were significantly higher in patients with skin tumors or erythroderma. CC genotype (rs2910164 C>G) was significantly more frequent in healthy controls and associated with lower MF risk and lower miR-146a levels. The AA genotype (rs767649 T>A) was significantly more frequent in patients and correlated with increased MF risk and increased miR-155 levels. The combination of GG+AA was only detected in patients and was correlated with higher MF susceptibility. Increased mir-146a and mir-155 plasma levels in MF is an important finding to establish putative noninvasive biomarkers. The presence of SNPs is closely associated with miRs' expression, and possibly with disease susceptibility.

Protein and mRNA Expression Levels of Interleukin-17A, -17F and -22 in Blood and Skin Samples of Patients with Mycosis Fungoides.

This study investigated the expression of interleukin (IL)-17A, -17F and -22 in mycosis fungoides. Blood samples were collected from 50 patients with mycosis fungoides and 50 healthy controls. Skin samples were obtained from 26 patients with mycosis fungoides and 5 healthy controls. Protein levels of IL-17A, -17F and -22 were measured in serum by multiplex enzyme-linked immunosorbent assay, and mRNA expression levels were measured in blood and skin samples by real-time quantitative reverse transcription PCR. Both IL-17A and IL-17F mRNA expression levels were significantly lower in blood of patients with mycosis fungoides in comparison with healthy controls. IL-22 serum levels and expression levels of IL-22 mRNA in skin tissue, were significantly increased in patients with mycosis fungoides in comparison with healthy controls. These results suggest that low levels of IL-17A and IL-17F in mycosis fungoides may be connected to impaired immune surveillance contributing to tumourigenesis. Upregulation of IL-22 may play a role in the establishment of the tumour microenvironment in mycosis fungoides.

Bilateral Aleukemic Myeloid Sarcoma of the Eyelids With Indolent Course.

Leukemic infiltrates may be seen in the skin in the absence of detectable bone marrow involvement. Leukemia cutis may exceptionally occupy the eyelids. An unusual case of a 58-year-old man presenting bilateral erythematous eyelid lesions, proven to be aleukemic leukemia cutis, is reported. Biopsy was conducted and hematoxylin/eosin stained sections were histologically evaluated. Immunohistochemistry was also performed.Light microscopy revealed cutaneous infiltration by a neoplastic population consisting of medium-sized cells. These cells infiltrated the overlying epidermis leading to focal microulcerations. The morphological and immunohistochemical characteristics of the neoplastic population were compatible with myeloid leukemia cutis. The bone marrow biopsy was normocellular for the patient's age. Although chemotherapy was advised, the patient refused any treatment. He remains free of leukemia or evolution of eyelid lesions approximately 1 year after diagnosis. Leukemia cutis of the eyelids is a rare manifestation of acute leukemia and may remain aleukemic in adults for an indefinite period of time.

miRNAs as predictive biomarkers of response to treatment in pediatric patients with acute lymphoblastic leukemia.

MicroRNAs (miRNAs/miRs) are promising prognostic biomarkers in pediatric acute lymphoblastic leukemia (ALL). The present study aimed to identify miRNAs that could serve as prognostic biomarkers or as novel therapeutic targets in ALL. The expression levels of 84 miRNAs were assessed in the bone marrow aspirates of 10 pediatric patients with newly diagnosed ALL at diagnosis and on day 33 of induction of the ALL Intercontinental Berlin-Frankfurt-Münster 2009 protocol, and associations with established prognostic factors were evaluated. The levels at diagnosis of 25 miRNAs were associated with ≥2 prognostic factors. Higher expression levels of let-7c-5p, miR-106b-5p, miR-26a-5p, miR-155-5p, miR-191-5p, miR-30b-5p and miR-31-5p were significantly associated with a good prednisone response. The expression levels of miR-125b-5p, miR-150-5p and miR-99a-5p were significantly higher in standard- or intermediate-risk patients compared with those in high-risk patients (P=0.017, P=0.033 and P=0.017, respectively), as well as in those with a complete response at the end of induction (P=0.044 for all three miRNAs). The change in expression levels between diagnosis and the end of induction differed significantly between risk groups for three miRNAs: miR-206, miR-210 and miR-99a (P=0.033, P=0.047 and P=0.008, respectively), with the post induction levels of miR-206 increased in high-risk patients, whilst miR-210 and miR-99a levels were increased in intermediate/standard risk patients. Therefore, miRNAs that could be integrated into the risk stratification of pediatric ALL after further evaluation in larger patient cohorts were identified.

Effect of apolipoprotein E (APOE) gene polymorphisms on the lipid profile of children being treated for acute lymphoblastic leukemia.

BACKGROUND: Medications used to treat acute lymphoblastic leukemia (ALL), such as L-asparaginase, can cause blood lipid disturbances. These can also be associated with polymorphisms of the lipoprotein lipase (LpL) and apolipoprotein E (APOE) genes. PROCEDURE: We aimed to investigate the association between lipid profile, certain LpL and APOE gene polymorphisms (rs268, rs328, rs1801177 and rs7412, rs429358 respectively) as well as the risk subgroup in 30 pediatric patients being treated for ALL, compared with 30 pediatric ALL survivors and 30 healthy controls. RESULTS: The only APOE gene polymorphism with significant allelic and genotypic heterogeneity was rs429358. Further analysis of this polymorphism showed that genotype (CC, CT, or TT) was significantly associated with (1) changes in the lipid profile at the end of consolidation (total cholesterol, LDL, apo-B100, and lipoprotein a) and during re-induction (total cholesterol and apo-B100), and (2) classification in the high risk-ALL subgroup (for CC genotype/C allele presence). CONCLUSIONS: Lipid abnormalities in children being treated for ALL may be associated with the APOE genotype, which is also possibly associated with risk stratification. Further research is needed to confirm the potential prognostic value of these findings.

Immunohistochemical Evidence Linking Interleukin-22 Tissue Expression Levels to FOXP3+ Cells and Neutrophil Densities in the Mycosis Fungoides Microenvironment.

BACKGROUND: Emerging data indicate that the cellular microenvironment and interleukins (IL) play a crucial role in mycosis fungoides (MF). We aimed to explore the potential association between the composition of the cellular microenvironment and the expression of IL-22 and IL-17A in MF skin lesions. METHODS: The study encompassed 16 cases of MF of different stages, for which sufficient skin tissue for immunohistochemistry and frozen tissue for reverse transcription-polymerase chain reaction, both taken from the same lesion, were available. Histological evaluation of eosinophils, neutrophils, CD20+, CD4+, CD8+, FOXP3+, CD56+, and CD1a+ cells was conducted. Additionally, mRNA expression levels of IL-22 and IL-17 mRNA were quantified using reverse transcription-quantitative polymerase chain reaction. SPSS version 28 (IBM Corp., Armonk, NY) was utilized for statistical analysis. RESULTS: Among the cases examined, three were in the patch stage, eight in the plaque stage, and five in the transformation to high-grade large cell lymphoma (t-LCL). B-lymphocytes, neutrophils, and eosinophils were primarily observed in t-LCL cases. IL-22 levels displayed a significant association with IL-17A levels (Pearson's r = 0.961, p < 0.001), FOXP3+ cells (Pearson's r = 0.851, p < 0.001), and neutrophil density (Pearson's r = 0.586, p = 0.014). No correlation was detected between IL-17A levels and the evaluated subtypes of microenvironmental cells. CONCLUSION: The microenvironment of MF lesions with t-LCL is noticeably different from early MF in terms of cellular composition. Histopathological identification of the cellular microenvironment may serve as an indicator of IL-22 tissue levels. These results implicate certain types of cells in IL-22 expression in the MF microenvironment and may contribute to advancing our knowledge on the pathogenesis and progression of the disease.

Efficacy and safety of mydriatic microdrops for retinopathy of prematurity screening: an external pilot crossover randomized controlled trial.

OBJECTIVE: To study the efficacy and safety of mydriatic microdrops compared with standard drops for retinopathy of prematurity (ROP) screening. STUDY DESIGN: Preterm infants undergoing ROP screening received microdrops and standard drops of phenylephrine 1.67% and tropicamide 0.33% in a random allocation sequence at two consecutive weekly examinations. Primary outcome was pupil diameter measured by two masked observers at 45 (T45) and 90 (T90) minutes after instillation. RESULTS: Twenty-five infants were randomized. No differences observed in mean pupil diameter after either administration technique at all time points (T45 Mean Difference: -0.14; 95% Confidence Interval: -0.38, 0.09; p = 0.23). Heart rate values at T120 were lower after microdrop instillation (p = 0.046). Otherwise, adverse events did not differ after either administration technique. CONCLUSION: This pilot study provides evidence of microdrops mydriasis efficacy, while justifying a full-scale trial to confirm their non-inferiority compared with standard drops and provide more data about safety. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04623684.

Evidence of somatic hypermutation in the antigen binding sites of patients with CLL harboring IGHV genes with 100% germline identity.

Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.

Male breast involvement in chronic lymphocytic leukemia. A case report and review of the literature.

Male breast lymphoma is a rare extranodal lymphoma occupying the mammary gland, and it could be either primary or secondary. A 78-year-old man presented an enlargement of the right breast. He had no medical history of interest. On physical examination, a unilateral, painless breast lump was found, with no skin changes or nipple discharge. There was no palpable lymphadenopathy. Routine laboratory tests revealed leukocytosis and lymphocytosis. Excisional biopsy of the breast lesion revealed mammary tissue infiltration by chronic lymphocytic leukemia (CLL) with plasmacytoid features and immunoglobulin G∕kappa monotypic expression. To our knowledge, this is the first report of male breast involvement by CLL. Considering important the collection of clinicopathological data of all reported male breast lymphoma cases, a literature review is presented.

MeinteR: A framework to prioritize DNA methylation aberrations based on conformational and cis-regulatory element enrichment.

DNA methylation studies have been reformed with the advent of single-base resolution arrays and bisulfite sequencing methods, enabling deeper investigation of methylation-mediated mechanisms. In addition to these advancements, numerous bioinformatics tools address important computational challenges, covering DNA methylation calling up to multi-modal interpretative analyses. However, contrary to the analytical frameworks that detect driver mutational signatures, the identification of putatively actionable epigenetic events remains an unmet need. The present work describes a novel computational framework, called MeinteR, that prioritizes critical DNA methylation events based on the following hypothesis: critical aberrations of DNA methylation more likely occur on a genomic substrate that is enriched in cis-acting regulatory elements with distinct structural characteristics, rather than in genomic "deserts". In this context, the framework incorporates functional cis-elements, e.g. transcription factor binding sites, tentative splice sites, as well as conformational features, such as G-quadruplexes and palindromes, to identify critical epigenetic aberrations with potential implications on transcriptional regulation. The evaluation on multiple, public cancer datasets revealed significant associations between the highest-ranking loci with gene expression and known driver genes, enabling for the first time the computational identification of high impact epigenetic changes based on high-throughput DNA methylation data.

Age-dependent methylation in epigenetic clock CpGs is associated with G-quadruplex, co-transcriptionally formed RNA structures and tentative splice sites.

Horvath's epigenetic clock consists of 353 CpGs whose methylation levels can accurately predict the age of individuals. Using bioinformatics analysis, we investigated the conformation, energy characteristics and presence of tentative splice sites of the sequences surrounding the epigenetic clock CpGs, in relation to the median methylation changes in different ages, the presence of CpG islands and their position in genes. Common characteristics in the 100 nt sequences surrounding the epigenetic clock CpGs are G-quadruplexes and/or tentative splice site motifs. Median methylation increases significantly in sequences which adopt less stable structures during transcription. Methylation is higher when CpGs overlap with G-quadruplexes than when they precede them. Median methylation in epigenetic clock CpGs is higher in sequences expressed as single products rather than in multiple products and those containing single donors and multiple acceptors. Age-related methylation variation is significant in sequences without G-quadruplexes, particularly those producing low stability nascent RNA and those with splice sites. CpGs in sequences close to transcription start sites and those which are possibly never expressed (hypothetical proteins) undergo similar extent of age-related median methylation decrease and increase. Preservation of methylation is observed in CpG islands without G-quadruplexes, contrary to CpGs far from CpG islands (open sea). Sequences containing G-quadruplexes and RNA pseudoknots, determining the recognition by H3K27 histone methyltransferase, are hypomethylated. The presented structural DNA and co-transcriptional RNA analysis of epigenetic clock sequences, foreshadows the association of age-related methylation changes with the principle biological processes of DNA and histone methylation, splicing and chromatin silencing.

Aberrant p16 promoter methylation among Greek lung cancer patients and smokers: correlation with smoking.

Genetic and environmental factors (dietary and smoking) influence lung cancer epidemiology and induce epigenetic modifications that should be assessed in individual populations. We analyzed p16 methylation among Greek non-small cell lung carcinoma patients and smokers using two-stage methylation-specific polymerase chain reaction. One hundred and fifty specimens from cancerous and adjacent non-cancerous tissue, bronchial washings and sputum from patients and 48 specimens, mostly sputum, from disease-free smokers were included. p16 methylation was very frequent in biopsies (82.85%) and bronchial washings (non-small cell lung carcinoma, 80.35%; small cell lung carcinoma, 16.66%) from patients, but also in adjacent non-cancerous tissue (45.71%). Concordance of p16 methylation and positivity by cytological examination was 51.78%. Methylation was also observed in sputum from asymptomatic cytology-negative smokers (22.5%) and chronic obstructive pulmonary disease patients (three of eight). Among disease-free individuals, methylation correlated only with heavy smoking (>50 pack-years, P<0.001) and differed among male and female disease-free smokers. In summary, p16 methylation is very frequent among non-small cell lung carcinoma patients, and correlates with heavy cigarette consumption only in disease-free smokers.

Association of two synonymous splicing-associated CpG single nucleotide polymorphisms in calpain 10 and solute carrier family 2 member 2 with type 2 diabetes.

Coding synonymous single nucleotide polymorphisms (SNPs) have attracted little attention until recently. However, such SNPs located in epigenetic, CpG sites modifying exonic splicing enhancers (ESEs) can be informative with regards to the recently verified association of intragenic methylation and splicing. The present study describes the association of type 2 diabetes (T2D) with the exonic, synonymous, epigenetic SNPs, rs3749166 in calpain 10 (CAPN10) glucose transporter (GLUT4) translocator and rs5404 in solute carrier family 2, member 2 (SLC2A2), also termed GLUT2, which, according to prior bioinformatic analysis, strongly modify the splicing potential of glucose transport-associated genes. Previous association studies reveal that only rs5404 exhibits a strong negative T2D association, while data on the CAPN10 polymorphism are contradictory. In the present study DNA from blood samples of 99 Greek non-diabetic control subjects and 71 T2D patients was analyzed. In addition, relevant publicly available cases (40) resulting from examination of 110 Personal Genome Project data files were analyzed. The frequency of the rs3749166 A allele, was similar in the patients and non-diabetic control subjects. However, AG heterozygotes were more frequent among patients (73.24% for Greek patients and 54.55% for corresponding non-diabetic control subjects; P=0.0262; total cases, 52.99 and 75.00%, respectively; P=0.0039). The rs5404 T allele was only observed in CT heterozygotes (Greek non-diabetic control subjects, 39.39% and Greek patients, 22.54%; P=0.0205; total cases, 34.69 and 21.28%, respectively; P=0.0258). Notably, only one genotype, heterozygous AG/CC, was T2D-associated (Greek non-diabetic control subjects, 29.29% and Greek patients, 56.33%; P=0.004; total cases, 32.84 and 56.58%, respectively; P=0.0008). Furthermore, AG/CC was strongly associated with very high (≥8.5%) glycosylated plasma hemoglobin levels among patients (P=0.0002 for all cases). These results reveal the complex heterozygotic SNP association with T2D, and indicate possible synergies of these epigenetic, splicing-regulatory, synonymous SNPs, which modify the splicing potential of two alternative glucose transport-associated genes.

The cellular microenvironment and neoplastic population in mycosis fungoides skin lesions: a clinicopathological correlation.

The cellular microenvironment has been proven to play a crucial role in solid tumours and seems to be important in haematologic malignancies, however, it has not been adequately investigated in primary cutaneous T cell lymphomas. The aim of this study was to register the composition of the cellular microenvironment in mycosis fungoides skin lesions and correlate the composing parameters with the clinical data and follow-up results. The presence of eosinophilic polymorphonuclear leukocytes, B lymphocytes, CD68+ macrophages, and CD1a+ epidermal Langerhans and antigen-presenting dermal dendritic cells, as well as their relation to clinicopathological parameters, were studied in 16 mycosis fungoides cases of different disease stages. The presence and nature of the participating T cell populations was also investigated. CD8+ tumour infiltrating T cells and CD56+ cells were found among neoplastic CD4+ T cells in the lesions. Generally, eosinophils and B lymphocytes were absent or in low numbers, regardless of clinical presentation, contrary to tumourous lesions. Macrophages and CD1a+ cells were constantly present, even in early-stage mycosis fungoides. The reduced presence of the CD1a+ population was associated with resistance to therapy (x2; p = 0.012). There is a striking difference in cellular microenvironment composition between early and advanced mycosis fungoides lesions.

Non-CpG cytosine methylation of p53 exon 5 in non-small cell lung carcinoma.

Non-CpG methylation of cytosine residues, a mechanism associated with regulation of gene expression, has not been investigated in human cancer until now. Analysis of the p53 exon 5 mutation spectrum in mutation databases for lung cancer reveals frequent GC>AT transitions, several of which occur at non-CpG sequences. To investigate the involvement of cytosine methylation in this mutagenesis process, we analyzed the methylation profile of p53 exon 5, in lung carcinoma. In this report, we present evidence that extensive clustered non-CpG methylation is observed in three regions of this exon, namely the sequences spanning codons 156-159, 175-179 and the 3' splice site, as well as in scattered CpA sequences. This methylation pattern was verified using direct methylation sequencing, and a two-stage methylation-specific PCR assay (MSP), designed for the detection of methylation in a GC rich region (oligo C sequence, of codons 175-179) of exon 5. The results from this MSP assay reveal that DNA from cancerous specimens was more heavily methylated in non-CpG cytosines, compared to that from non-cancerous lung tissue of cancer patients (14/19 cancerous and 6/19 non-cancerous, respectively). DNA isolated from human leucocytes and some non-cancerous specimens (2/19) was free of non-CpG methylation. Careful analysis of the mutations reported in p53 mutation databases also provides corroborating evidence that the high incidence of GC>AT mutations in the p53 gene, observed in lung cancer, might also be related to non-CpG methylation, as well as to the overall increase of methylation sites in this locus.