LncRNAs expression profile in a family household cluster of COVID-19 patients.

More than 3 years after the start of SARS-CoV-2 pandemic, the molecular mechanisms behind the viral pathogenesis are still not completely understood. Long non-coding RNAs (lncRNAs), well-known players in viral infections, can represent prime candidates for patients' risk stratification. The purpose of the current study was to investigate the lncRNA profile in a family cluster of COVID-19 cases with different disease progression, during the initial wave of the pandemic and to evaluate their potential as biomarkers for COVID-19 evolution. LncRNA expression was investigated in nasopharyngeal swabs routinely collected for diagnosis. Distinct expression patterns of five lncRNAs (HOTAIR, HOTAIRM1, TMEVPG1, NDM29 and snaR) were identified in all the investigated cases, and they were associated with disease severity. Additionally, a significant increase in the expression of GAS5-family and ZFAS1 lncRNAs, which target factors involved in the inflammatory response, was observed in the sample collected from the patient with the most severe disease progression. An lncRNA prognostic signature was defined, opening up novel research avenues in understanding the interactions between lncRNAs and SARS-CoV-2.

Assessment of Total Antioxidant Capacity, 8-Hydroxy-2'-deoxy-guanosine, the Genetic Landscape, and Their Associations in BCR::ABL-1-Negative Chronic and Blast Phase Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders defined by an excessive production of functionally mature and terminally differentiated myeloid cells. MPNs can transform into secondary acute myeloid leukemia (sAML/blast phase MPN) and are linked to alterations in the redox balance, i.e., elevated concentrations of reactive oxygen species and markers of oxidative stress (OS), and changes in antioxidant systems. We evaluated OS in 117 chronic phase MPNs and 21 sAML cases versus controls by measuring total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxy-guanosine (8-OHdG) concentrations. TAC was higher in MPNs than controls (p = 0.03), particularly in ET (p = 0.04) and PMF (p = 0.01). MPL W515L-positive MPNs had higher TAC than controls (p = 0.002) and triple-negative MPNs (p = 0.01). PMF patients who had treatment expressed lower TAC than therapy-free subjects (p = 0.03). 8-OHdG concentrations were similar between controls and MPNs, controls and sAML, and MPNs and sAML. We noted associations between TAC and MPNs (OR = 1.82; p = 0.05), i.e., ET (OR = 2.36; p = 0.03) and PMF (OR = 2.11; p = 0.03), but not sAML. 8-OHdG concentrations were not associated with MPNs (OR = 1.73; p = 0.62) or sAML (OR = 1.89; p = 0.49). In conclusion, we detected redox imbalances in MPNs based on disease subtype, driver mutations, and treatment history.

Kinetics and persistence of cellular and humoral immune responses to SARS-CoV-2 vaccine in healthcare workers with or without prior COVID-19.

SARS-CoV-2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS-CoV-2 infection. No new infections were diagnosed during follow-up. At 6 months, post-vaccination or post-infection, despite a downward trend in the level of anti-S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live-virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow-up in persons vaccinated after prior infection. Anti-S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1-3) or low neutralizing activity (30%-40%) at 6 months, although with lower T-cell stimulation index (p = 0.046) and IFN-γ secretion (p = 0.0007) compared to those with preserved humoral responses.

FACTORS ASSOCIATED WITH PERSISTENCE OF HPV GENITAL INFECTION IN A SMALL COHORT OF ROMANIAN WOMEN.

The aim of the study was to assess the role of behavioral factors in persistence of human papillomavirus (HPV) genital infection. Out of a cohort of 605 women included in a study of HPV infection prevalence, 142 HPV positive women (aged 18-57) were retested after a 12-month interval. None of the patients underwent surgical treatment during that period. Selected patients were asked for a second smear for cytologic analysis and HPV genotyping. A questionnaire that included information regarding reproductive health, sexual activity and smoking status was filled-in. After 12 months, 46 of 142 (32.39%) women had persistent HPV infection, with genotypes 16 and 18 found in 27 cases. On the other hand, 17 of 142 (11.97%) women had acquired new infections replacing the baseline genotypes. In our study, smoking (OR=2.320, p=0.0330) and sexual behavior (OR=5.333, p=0.0180 for more than three sexual partners; OR=2.427, p=0.0238 for cases where the partner was involved in another sexual relationship) were associated with viral persistence, while long-term contraception did not yield statistically significant results.

LINC01101 and LINC00277 expression levels as novel factors in HPV-induced cervical neoplasia.

Recently long non-coding RNAs were identified as new factors involved in gene expression regulation. To gain insight into expression pattern of these factors related to E7 HPV18 oncogene, this study uses HeLa cell culture transfected with E7-siRNA. Gene expression profile was investigated using microarray analysis. After analysing the microarray results, we identified 15,387 RNA species differentially expressed in E7-siRNA-transfected cells compared with controls (fold change >2). The expression profiles of lncRNA species highlighted 731 lncRNAs and 203 lincRNAs. We selected two lincRNAs (LINC01101 and LINC00277) and we evaluated the expression profile in HPV-induced neoplasia. Both lincRNAs investigated display a significantly reduced pattern of expression in cervical lesions and cancer, associated with clinical parameters. A connection between HPV presence and lincRNAs was noted. hrHPV-positive samples exhibit significantly reduced LINC01101 and LINC00277 expression level (P < 0.05). These results provide new insights into involvement of lncRNA in HPV-induced cervical cancer, enriching our understanding of their potential role in this pathology.

miR-29a associates with viro-immunological markers of HIV infection in treatment experienced patients.

MicroRNAs (miRNAs) are small, non-coding RNA species essential for the post-translational regulation of gene expression. Several miRNA have been proposed to contribute to Human immunodeficiency virus-1 (HIV-1) infection establishment, progression and latency. Among them, miR-29a seems to be of particular interest. The aim of this study was to investigate the association between miR-29a expression and immunologic and virologic markers of HIV infection progression in long-term antiretroviral-treated individuals. In a homogenous group of 165 young adults, with chronic HIV infection, parenterally acquired during childhood, the expression level of miR-29a was found to be inversely correlated with HIV viral load and the degree of immunosuppression, expressed by both CD4 cell count and the CD4/CD8 ratio. There was a significant difference in miR-29a expression according to the patient's response to treatment, with the lowest levels expressed by patients with treatment failure, defined as detectable viremia and CD4 < 350 cells/mm3 . No significant correlation was found between miRNA level and the nadir CD4 count or zenith HIV viral load. This study establishes the association between miR-29a expression and markers of HIV infection in long-term survivors, treatment-experienced patients, suggesting its potential use as an indicator for the on-treatment disease evolution. J. Med. Virol. 88:2132-2137, 2016. © 2016 Wiley Periodicals, Inc.

E5HPV16 mRNA EXPRESSION PATTERN ANALYSIS IN PATIENTS WITH CERVICAL LESIONS IN VIRAL STATUS CONTEXT.

Human papilloma virus (HPV) may cause mostly transient infections of cutaneous and mucous epithelia. Persistent HPV genital infections may induce pre-malignant or malignant lesions. While E6 and E7 HPV genes' malignant character is known, E5 is still under debate. We evaluated the possible role of E5 gene in cervix oncogenesis, in patients with abnormal cytology and HPV1 6 positive, in the context of viral status correlated with potential targets (p21, EGFR). HPV DNA was detected and genotyped using Linear Array HPV Genotyping Test (Roche Molecular Biochemicals, Mannheim, Germany) and E2, E6, E5 HPV16, p21 and EGFR transcripts levels were investigated by qRT-PCR. Our results indicate a significantly high E5 expression in low grade cytology, expression correlated with a moderated E6 and low p21 levels. All HSIL specimens presented integrated/mixed viral forms; mixed forms presented moderate E5 expression, high levels of p21 correlates with E6 oncogene high expression. These findings indicate a potential role for E5 pattern of expression in discriminating be-tween lesions that may progress to cancer.

THE PERFORMANCE OF A RAPID TEST FOR ANTI-HCV SCREENING IN ORAL FLUIDS.

UNLABELLED: Gingival crevicular fluid (GCF) and saliva samples provide advantages for screening or sero-prevalence studies on HCV using less invasive methods. The study aimed to evaluate the performance of a rapid test for HCV-antibodies (HCV-Ab) screening in oral fluids among high-risk individuals with chronic liver disease. METHODS: Chronic liver disease patients attending at the Matei Bals National Instiute for Infectious Diseases were recruited for this study. Plasma, GCF and saliva samples (pair samples) were collected from each patient included in the study. Forty-three sample pairs were tested with Laboquick (Koroglu Medical Devices) rapid test and ELISA (DIA.PRO--Diagnostic Bio-probes) for the detection of anti-HCV antibodies. RESULTS: Using rapid test, anti-HCV antibodies were detected in 36 GCFs (83.72%) and 24 saliva cases (55.8%) of infected subjects. For a better estimation of oral fluids positivity, the cut-off values were calculated following plotting the ROC curves (COV2). Comparing Laboquick and ELISA (COV2) data, matched results were noted in 95.3 % saliva samples and 93% GCF samples. CONCLUSIONS: Oral fluids could be an alternative to blood for detection of HCV-positive subjects. Anti-HCV rapid test may be useful in routine dental medicine.

Methylation pattern of methylene tetrahydrofolate reductase and small nuclear ribonucleoprotein polypeptide N promoters in oligoasthenospermia: a case-control study.

Alterations in DNA methylation patterns in several genes may lead to abnormal male sexual development and infertility. This study investigated the promoter methylation status of MTHFR and SNRPN in infertile men from Romania by quantitative methylation-specific PCR in order to investigate possible correlations with sperm abnormalities. The study groups included patients (n=27) with a median age of 31 years (range 26-41 years) as well as controls (n=11) with a median age of 30 years (range 24-37 years) recruited from couples seeking advice for infertility. DNA was isolated from sperm samples and promoter methylation was assessed using direct. Significant trends were detected for both genes that indicate a tendency towards promoter hypermethylation in spermatozoa with low motility (MTHFR P=0.0032, r=0.23; SNRPN P=0.0003, r=0.32) and poor morphology (MTHFR P=0.0012, r=0.27; SNRPN P=0.0003, r=0.33) but no trend was found in cases of low sperm count (MTHFR r=0.007; SNRPN r=0.06). The data indicate that the methylation patterns of the promoters of MTHFR and SNRPN are associated with changes in sperm motility and morphology, which could lead to male infertility. A large number of studies are now focused on the causes of male infertility. Among these are epigenetic modifications, which are important contributors to reproductive pathology in the male by providing dynamic changes of the phenotype according to the environmental and metabolic factors. The most known epigenetic modification is DNA methylation and alterations in this pattern in several genes could induce male infertility. The present study aims to investigate the promoter methylation status of the genes for methylene tetrahydrofolate reductase (MTHFR) and small nuclear ribonucleoprotein polypeptide N (SNRPN) in infertile males from Romania, in order to establish a correlation with sperm parameters. MTHFR is an enzyme involved in the folate pathway and in de novo nucleotide biosynthesis but also a good example for gene-environment interaction in phenotype development. SNRPN is involved in both somatic cell expression and inheritance of the imprint and the methylation pattern of its gene seems to correlate not only with imprinted disorders but also with infertility. Our study includes patients (n=27, median age 31 years, range 26-41 years) recruited from men seeking advice for couple infertility and control group (n=11, median age 30.5 years, range 24-37 years). The data we obtained indicated significant correlations between hypermethylation of the investigated genes and sperm motility and morphology. No significant correlation between DNA methylation and sperm number was found. Our data suggest that methylation pattern of MTHFR and SNRPN is linked with sperm anomalies of motility and morphology and therefore male infertility.

Molecular variants of human papilloma virus 16 E2, E4, E5, E6 and E7 genes associated with cervical neoplasia in Romanian patients.

The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p < 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis.

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and promoter methylation in cervical oncogenic lesions and cancer.

The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens (n = 77)/tumour tissue specimens (n = 23) were investigated. As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk (normal/cervical lesions and tumours: wild homozygote 34/41 (0.68/0.41), heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV-negative and HPV-positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV-positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high-risk (hr)HPV versus low-risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV.

Exploring Microbiota Diversity in Cervical Lesion Progression and HPV Infection through 16S rRNA Gene Metagenomic Sequencing.

(1) Background: Cervical cancer is a significant health concern, with the main cause being persistent infection with high-risk Human Papillomavirus (hrHPV). There is still no evidence for why viral persistence occurs in some women, but recent studies have revealed the interplay between cervical microbiota and hrHPV. This research aimed to characterize the cervicovaginal microbiota in cervical lesion progression and HPV infection status. (2) Methods: This study included 85 cervical specimens from women from the north-eastern region of Romania. DNA was isolated from cervical secretion for HPV genotyping and 16S ribosomal RNA gene NGS sequencing. (3) Results: Our study revealed a distinct pattern within the studied group when considering Lactobacillus species, which differs from findings reported in other populations. Specifically, the presence of Lactobacillus iners coupled with the absence of Lactobacillus crispatus alongside Atopobium spp., Prevotella spp., and Gardnerella spp. could serve as defining factors for severe cervical lesions. The results also showed a significant association between microbiota diversity, HPV infection, and cervical lesion progression. (4) Conclusions: As the microbiota profile seems to vary among different populations and individuals, a deeper comprehension of its composition has the potential to develop personalized detection and treatment approaches for cervical dysplasia and cancer.

Leukemic conversion involving RAS mutations of type 1 CALR-mutated primary myelofibrosis in a patient treated for HCV cirrhosis: a case report.

Somatic frameshift mutations in exon 9 of calreticulin (CALR) gene are recognized as disease drivers in primary myelofibrosis (PMF), one of the three classical Philadelphia-negative myeloproliferative neoplasms (MPNs). Type 1/type 1-like CALR mutations particularly confer a favorable prognostic and survival advantage in PMF patients. We report an unusual case of PMF incidentally diagnosed in a 68-year-old woman known with hepatitis C virus (HCV) cirrhosis who developed a progressive painful splenomegaly, without anomalies in blood cell counts. While harboring a type 1 CALR mutation, the patient underwent a leukemic transformation in less than 1 year from diagnosis, with a lethal outcome. Analysis of paired DNA samples from chronic and leukemic phases by a targeted next-generation sequencing (NGS) panel and single-nucleotide polymorphism (SNP) microarray revealed that the leukemic clone developed from the CALR-mutated clone through the acquisition of genetic events in the RAS signaling pathway: an increased variant allele frequency of the germline NRAS Y64D mutation present in the chronic phase (via an acquired uniparental disomy of chromosome 1) and gaining NRAS G12D in the blast phase. SNP microarray analysis showed five clinically significant copy number losses at regions 7q22.1, 8q11.1-q11.21, 10p12.1-p11.22, 11p14.1-p11.2, and Xp11.4, revealing a complex karyotype already in the chronic phase. We discuss how additional mutations, detected by NGS, as well as HCV infection and antiviral therapy, might have negatively impacted this type 1 CALR-mutated PMF. We suggest that larger studies are required to determine if more careful monitoring would be needed in MPN patients also carrying HCV and receiving anti-HCV treatment.

Mitochondrial DNA mutations in patients with HRHPV-related cervical lesions.

High risk human papillomaviruses (hr-HPV) are known to be the etiological agents of cervical cancer disease. On the other hand, other cofactors are considered to be important in cervix carcinogenesis. Mutations in mitochondrial DNA (mtDNA) as well as alterations in mtDNA content have been reported in numerous cancers examined to date. The D-loop region has been shown to be a mutational "hot spot" in human cancer. In order to evaluate the role of mtDNA mutations in cervical lesions progression, cervical specimens (from 79 women, 29-65 years old) were investigated. DNA was isolated (High Pure PCR Template, Roche Diagnostics) from cervical cells from patients with different cytology (normal cervical epithelium, ASCUS-Atypical Squamous Cells of Undetermined Significance, LGSIL-Low-Grade Intraepithelial Lesion, HGSIL-High-Grade Intraepithelial Lesion and SCC-Squamous Cell Carcinoma) and tested for HPV DNA presence (Linear Array HPV Genotyping Test, Roche Diagnostics). To elucidate a causative role of mtDNA in cervical lesions, mtDNA mutations were investigated using Mutector mtDNA kit (TrimGen Corporation). In patients with normal and ASCUS cytology, mtDNA mutations were absent. 16.66% of LGSIL patients presented mutations in D-loop region whereas 28.57% HGSIL cases showed mutations in mtDNA. Mutations were detected in 66.66% cases of SCC cases. These studies provide strong evidence that instability in the D-loop region of mtDNA may be involved in cervical dysplasia. We suggested that mtDNA mutations may play a role in cervical precursor lesions and cancer but their role in the mechanism of carcinogenesis remains to be solved.

Epigenetic approaches for cervical neoplasia screening (Review).

Human papillomavirus (HPV) infection is the leading cause of cervical cancer. The Papanicolaou cytology test is the usually employed type of screening for this infection; however, its sensibility is limited. Only a small percentage of women infected with high-risk HPV develop cervical cancer with an array of genetic and epigenetic modifications. Thus, it is necessary to develop rapid, reproducible and minimally invasive technologies for screening. DNA methylation has gained attention as an alternative method for molecular diagnosis and prognosis in HPV infection. The aim of the present review was to highlight the potential of DNA methylation in cervical neoplasia screening for clinical applications. It was observed that the methylation human and viral genes was correlated with high-grade lesions and cancer. Methylation biomarkers have shown a good capacity to discriminate between high-grade lesions with a transformative potential and cervical cancer, being able to detect these modifications at an early stage. With further research, the epigenetic profiles and subtypes of the tumors could be elaborated, which would aid in therapy selection by opening avenues in personalized precision medicine. Response to therapy could also be evaluated through such methods and the accessibility of liquid biopsies would allow a constant monitoring of the patient's status without invasive sampling techniques.

The association between lncRNA H19 and EZH2 expression in patients with EBV-positive laryngeal carcinoma.

OBJECTIVE: Laryngeal cancer is the second most common malignancy in the head and neck, with Epstein-Barr virus infection as a risk factor. Our aim is to evaluate correlations between the expression of lncRNA H19 and EBV infection in laryngeal cancer and H19 involvement in neoplastic progression through EZH2 association. MATERIALS AND METHODS: 30 paired laryngeal tissue specimens (neoplastic and non-neoplastic) were included in the study. Nucleic acid isolation and cDNA synthesis was performed according to the manufacturer's protocol. EBV DNA and expression of lytic (BZLF1) and latent (LMP1) forms of infection were assessed in PCR assays; expression levels of H19 and EZH2 were quantified in qRT-PCR. Data was analysed using GraphPad Prism 5.0. RESULTS: Higher H19 relative expression in neoplastic vs paired non-neoplastic samples was found (p < 0.0001) with a significant increase in EBV DNA positive neoplasms (p = 0.0434). An inverse correlation between H19 and EZH2 expression levels was noticed in EBV positive cases. Additionally, increased levels of H19 in LMP1 positive samples compared with those positive for BZLF1 was found (p = 0.0593). CONCLUSIONS: lncRNA H19 and EZH2 significantly contribute to the development of laryngeal carcinoma, being correlated with EBV infection markers.

TGF-beta signalling pathway factors in HPV-induced cervical lesions.

Human papillomaviruses (HPV) are considered the etiological agents of cervical cancer, especially high-risk genotypes. TGF-beta (transforming growth factor-beta) is well known for its anti-proliferative effects but the neoplastic cells often lose their sensitivity to TGF-beta. A characteristic alteration associated with malignant progression is the loss of responsiveness to TGF-beta1-induced cell growth inhibition. The aim of the present study was to establish the possible role of some members of TGF-beta signalling pathway during cervical cancer development and the possible relationship with HPV infection. In order to establish TGF-beta gene expression levels in cervical oncogenesis, TGF-beta1, TGF-beta1 receptors and Smad2 were investigated in precancerous and cervical cancer samples (Quantitative Real-Time PCR). The study revealed that 84.5% of patients were positive for HPV DNA. The most prevalent HPV genotypes were high-risk HPV 16 and 18 in single or co-infections. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia to cervical carcinoma. Furthermore, we observed that cervical lesions without HPV infection expressed significantly less TGF-beta1. TGF-betaRI and Smad2 gene expression levels were found to be decreased in SCC and AC samples in contrast with CIN1 and CIN2/3 samples. Our results showed that in human cervical cancer the disruption of TGF-beta/Smad signalling pathway might contribute to the malignant progression of cervical dysplasia. These data emphasize the importance of canonical TGF-beta pathway integrity in carcinogenesis.

Alterations of regulatory factors and DNA methylation pattern in thyroid cancer.

PURPOSE: DNA methylation plays an important role in thyroid oncogenesis. The aim of this study was to investigate the connection between global and local DNA methylation status and to establish the levels of important DNA methylation regulators (TET family and DNMT1) in thyroid tumours: follicular adenoma-FA, papillary thyroid carcinoma-PTC (classic papillary thyroid carcinoma-cPTC and papillary thyroid carcinoma follicular variant fvPTC). METHODS: Global DNA methylation profile in thyroid tumours tissue (41 paired samples) was assessed by 5-methylcytosine and 5-hydroxymethylcytosine levels evaluation (ELISA), along with TETs and DNMT1 genes expression quantification. Also, it was investigated for the first time TET1 and TET2 promoter's methylation in thyroid tumours. BRAF V600E mutation and RET/PTC translocation testing were performed on all investigated samples. In vitro studies upon DNA methylation in K1 thyroid cancer cells were performed with demethylating agents (5-AzaC and vitamin C). RESULTS: TET1 and TET2 displayed a significantly reduced gene expression level in PTC, while DNMT1 gene presented a high level of expression. PTC samples presented increased levels of 5-methylcytosine and low levels of 5-hydroxymethylcytosine. 5-methylcytosine levels were associated with TET1/TET2 expression levels. TET1 gene expression was significantly lower in patients positive for BRAF mutation and with RET/PTC rearrangement. TET2 gene was found hypermethylated in thyroid carcinoma patients overall, especially in PTC-follicular variant samples (p= 0.0002), where TET2 gene expression levels were significantly reduced (p= 0.0031). Furthermore, the data indicate for all thyroid cancer patients a good sensitivity (81.08%) and specificity (86.49%) regarding the use of TET1 (p< 0.0001), and TET2 (71.79%, 64.10%, p= 0.0001) hypermethylation as biomarkers for thyroid oncogenesis. CONCLUSIONS: These results suggest that TET1/TET2 gene expression and methylation may serve as potential diagnostic tools for thyroid neoplasia. Our study showed that the methylation of TET1 increases in malignant thyroid tumours. fvPTC patients presented lower methylation levels compared to cPTC and could be a discriminatory factor between two cancer types and benign lesions. TET2 is a poorer discriminator between FA and fvPTC, but it can be useful for cPTC identification. K1-cells treated with demethylating agents showed a demethylation effect, especially upon TET2 gene. The cumulative effect of L-AA and 5-AzaC proved to have a potent combined demethylating effect on genes promoter's activation and could open new perspectives for thyroid cancer therapy.

Single-Center Experience With Epigenetic Treatment for Juvenile Myelomonocytic Leukemia.

Background: Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm diagnosed in young children, characterized by somatic or germline mutations that lead to hyperactive RAS signaling. The only curative option is hematopoietic stem cell transplantation (HSCT). Recent data showing that aberrant DNA methylation plays a significant role in pathogenesis and correlates with clinical risk suggest a possible benefit of hypomethylating agents (HMA) in JMML treatment. Aim: The aim is to report the results of HMA-based therapy with 5-azacytidine (AZA) in three JMML patients treated in a single center, non-participating in EWOG-MDS study. Methods: The diagnosis and treatment response were evaluated according to international consensus criteria. AZA 75 mg/m2 intravenous (i.v.) was administered once daily on days 1-7 of each 28-day cycle. All patients were monitored for hematologic response, spleen size, and evolution of extramedullary disease. Targeted next generation sequencing (NGS) were performed after the 3rd AZA cycle and before SCT to evaluate the molecular alterations and genetic response. Results: Three patients diagnosed with JMML were treated with AZA (off-label indication) in Pediatric Department of Fundeni Clinical Institute, Bucharest, Romania between 2017 and 2019. There were two females and one male with median age 11 months, range 2-16 months. The cytogenetic analysis showed normal karyotype in all patients. Molecular analysis confirmed KRAS G13D mutation in two patients and NRAS G12D mutation in one patient. The clinical evaluation showed important splenomegaly and hepatomegaly in all 3 pts. One patient received AZA for early relapse after haploidentical HSCT and the other two patients received upfront AZA, as bridging therapy before HSCT. After HMA therapy, 2/3 patients achieved clinical partial response (cPR), 1/3 had clinical stable disease (cSD) and all had genetic stable disease (gSD) after 3 cycles and were able to receive the planned HSTC. One patient achieved clinical and genetic complete response before HSCT. During 22 cycles of AZA there were only four adverse events but only one determined dose reduction and treatment delay. Conclusion: Our data show that AZA monotherapy is safe and effective in controlling disease both in upfront and relapsed patients in order to proceed to HSCT.