Understanding the Pattern of Oropharyngeal Cancers from North-East Romanian Patients

Background: Human papilloma virus (HPV) is acknowledged as a risk factor for oropharyngeal squamous cellular cancers (OPSCC), of which the dominant types are tonsillar (TSCC) and base of tongue cancer (BOTSCC). Objective: To assess the role of HPV in selected OPSCC cases, from Romanian patients by sensitive and complementary molecular assays. Material and Methods: Fifty-four formalin fixed paraffin embedded (FFPE) OPSCC samples were analyzed for HPV DNA by a PCR-based bead-based multiplex-assay. Thirty-four samples were tested for HPV RNA and for overexpression of p16INK4a by immunohistochemistry. Twenty samples were evaluated by Competitive Allele-Specific Taqman PCR (CAST-PCR) for fibroblast growth factor receptor 3 protein (FGFR3) status. Results: A total of 33.3% (18/54) OPSCC samples were positive for HPV DNA. HPV16 was the most frequent type (30%, 16/54); followed by HPV18 (3.7%, 2/54); and 1 sample (1.8%) was positive for both HPV16 and 18. HPV18 E6*I was detected in a HPV18 DNA-positive oropharynx tumor. Four samples positive for HPV16 were also positive for p16INK4a. All the tested samples were negative for FGFR3. Conclusions: The increased HPV16 prevalence is in line with similar studies and is a new confirmation that HPV16 is the most prevalent type in our country; supporting the potential benefit of prophylactic vaccines. Overall, there is no concordance between DNA and any of the two other analytes that are considered being markers of HPV-driven cancers. There is a need to explore novel screening strategies that could be broadly used in the clinical routine to initiate preventive measures.

Study of the FUT2 gene association with infection and clinical manifestations of Helicobacter pylori infection

The aim of the pilot study of a group of adolescents with H. pylori infection was to study the preliminary data obtained on the rs602662 locus of the FUT2 gene and to establish its role in the realization of clinical manifestations of chronic gastritis, gastric ulcer and duodenal ulcer associated with H. pylori.Methods: The study included 91 patients. The study for the presence of the polymorphic locus rs602662 of the FUT2 gene was carried out by the standard TaqMan PCR method on a Real-Time CFX96 Touch amplifier. The duration of the study was 6 months.Results: The main group included 25 adolescents aged 16 to 17 years 11 months, the control group included 20 patients. Patients infected with H. pylori more often noticed symptoms of dyspepsia - in 36%, compared with the control group - 9.7%. The presence of a family history in the main group for associated diseases had a significant difference, χ2 = 4.97, p <0.05.To assess the contribution of the genotype of the rs602662 locus of the FUT2 gene to the risk of clinical manifestations in H. pylori infection, the main group was divided into subgroups. In the distribution of alleles in these groups, statistically significant differences were revealed.Allele “A” has a protective effect against the onset of clinical symptoms of dyspepsia. The odds ratio (OR) with the carriage of allele “A” (genotypes A / A and G / A versus G / G) to have clinical symptoms with a positive H. pylori status was 0.175 (CI = [0.049-0.625] chi2 = 7.79 p = 0.0053).Conclusion. As a result of the study, we were unable to identify a significant association of alleles and genotypes of the rs602662 locus of the FUT2 gene with clinical manifestations of H. pylori infection. At the same time, carriers of the A allele have a pronounced association with the absence of clinical symptoms in patients with a positive H. pylori infection status of 0.175 (C.I. = [0.049-0.625] chi2 = 7.79 p = 0.0053).

Beclometasone but not fluticasone modulates PDGFD expression in the H295R adrenal cell line.

BackgroundAdrenal suppression is a clinically concerning side effect of inhaled corticosteroid (ICS) treatment in patients with asthma. Increased susceptibility to ICS-induced adrenal suppression has previously been identified in those with the rs591118 polymorphism in Platelet Derived Growth Factor D (PDGFD). The mechanism underpinning this relationship is not known.MethodsH295R cells were genotyped for rs591118 using a validated Taqman PCR allelic discrimination assay. H295R cell viability was determined after treatment with beclometasone and fluticasone (range 0-330 μM). Cortisol was measured in cell culture medium using competitive enzyme immunoassay.ResultsPDGFD protein expression in H295R cells was confirmed using Western blotting. When ACTH and forskolin were added to H295R cells, a reduction in PDGFD expression was seen which was then restored by incubation with prochloraz, a known inhibitor of steroidogenesis.A dose-dependent, decrease in PDGFD expression was observed with beclometasone (over a 24 h incubation period) but not with beclometasone incubations beyond 24 hour nor with fluticasone (at 24 or 48 hours).ConclusionsH295R cells express PDGFD protein which can be modulated by incubation with steroidogenesis agonists and antagonists and additionally with exogenous beclometasone.

Development of Real-Time PCR Based on A137R Gene for the Detection of African Swine Fever Virus

African swine fever virus (ASFV) can infect domestic pigs and wild boars and causes huge economic losses in global swine industry. Therefore, early diagnosis of ASFV is important for the control and eradication of African swine fever (ASF). In this study, a SYBR Green-based real-time polymerase chain reaction (PCR) assay targeting the viral encoded A137R gene was established for the detection of ASFV infection. For the evaluation of the established real-time PCR, 34 clinical samples were assessed by both the A137R gene-based real-time PCR and OIE-recommended TaqMan PCR. The results showed that 85.29% (29/34) were detected by A137R gene-based real-time PCR, but only 79.41% (27/34) positive using OIE-recommended TaqMan PCR. Moreover, no cross-reaction with other common swine pathogens was found in the A137R gene-based real-time PCR. These results demonstrated that the established real-time PCR assay in this study showed better performance than the OIE-recommended method in detecting ASFV from clinical samples, which could be applied for control and eradication programs of ASF.

High Rate of Cytomegalovirus Detection in Cholestatic Preterm Infants

Objectives: To evaluate the prevalence of cytomegalovirus (CMV) infection in preterm infants with cholestasis.Study design: Preterm infants (&lt;37 weeks gestational age) with cholestasis were tested for CMV DNA using Taqman PCR in blood cells from sedimented whole blood, plasma, and urine. Infants were regarded as positive for CMV if any sample was tested positive. Their mothers were tested for CMV serostatus simultaneously. A control group of non-cholestatic preterm infants, and their mothers, were tested at a similar age.Results: A total of 69 preterm infants with a median gestational age of 26 weeks and 5 days were included, 45 cholestatic and 24 non-cholestatic. Of the cholestatic infants, 31/45 (69%) were CMV positive vs. 3/24 (13%) of the non-cholestatic infants (p &lt; 0.001). Cholestatic infants were equally preterm as the non-cholestatic ones, but were more severely ill. After adjusting for the risk factors necrotizing enterocolitis, prolonged parenteral nutrition, and gestational age, being CMV positive remained significantly associated with cholestasis in a multivariable logistic regression model. Characteristics of CMV-positive and -negative cholestatic infants showed differences only for necrotizing enterocolitis, occurring in 55% (17/31) of CMV positive vs. 21% (3/14) of CMV negative (p = 0.054), and mortality. Eight cholestatic CMV-positive infants died (26%) vs. none of the CMV-negative infants (p = 0.044).Conclusions: CMV DNA was detected in two out of three cholestatic preterm infants, by far more often than in the non-cholestatic control group. Cholestasis with simultaneous detection of CMV DNA may be associated with increased mortality.

Identification of a novel statovirus in a faecal sample from a calf with enteric disease

A novel clade of RNA viruses was identified in the mammalian gastrointestinal tract by next-generation sequencing. Phylogenetically, these viruses are related to the genera Tombusviridae (plant viruses) and Flaviviridae, which includes mammalian, avian and insect hosts. Named in line with their characterization as stool-associated Tombus-like viruses, it is unclear if statoviruses infect mammals or are dietary in origin. Here, metagenomic sequencing of faecal material collected from a 10-week-old calf with enteric disease found that 20 % of the reads mapped to a de novo-assembled 4 kb contig with homology to statoviruses. Phylogenetic analysis of the statovirus genome found a clear evolutionary relationship with statovirus A, but, with only 47 % similarity, we propose that the statovirus sequence presents a novel species, statovirus F. A TaqMan PCR targeting statovirus F performed on faecal material found a cycle threshold of 11, suggesting a high titre of virus shed from the calf with enteric disease. A collection of 48 samples from bovine enteric disease diagnostic submissions were assayed by PCR to investigate statovirus F prevalence and 6 of 48 (12.5 %) were positive. An ELISA to detect antibodies to the coat protein found that antibodies to statovirus F were almost ubiquitous in bovine serum. Combined, the PCR and ELISA results suggest that statovirus F commonly infects cattle. Further research is needed to elucidate the aetiological significance of statovirus infection.

Association study between asthma and single nucleotide polymorphisms of ORMDL3, GSDMB, and IL1RL1 genes in an Algerian population

Background Genetic variation has a key role in the development of asthma, but genetic influences may vary between different populations. In this study, we looked for evidence of association of key asthma SNPs, namely, rs1420101 and rs10192157 within the IL1RL1 gene, rs2305480 in GSDMB gene, and the rs3744246 polymorphism in the ORMDL3 gene, in the Algerian population. We included 266 unrelated subjects of an Algerian population in a case-control study, with 125 adult asthmatic and 141 healthy controls. DNA was extracted and genotypes determined by the Taqman PCR technique for characterization of the different genetic variants. Results The results show that there were no significant differences in allele frequencies for 3 of the chosen SNPs in the ORMDL3, GSDMB, and IL1RL1 genes between the asthmatic and control groups with respective P values of 0.922, 0.331, and 0.937. However the T allele of rs10192157 of the IL1RL1gene was associated with protection from asthma (P value=0.010). Conclusion These results indicate that there is no marked effect of rs3744246, rs2305480, and rs1420101 polymorphisms of the ORMDL3, GSDMB, and IL1RL1 genes on asthma risk in the Algerian population. However, a protective effect of the rs10192157 polymorphism of the IL1RL1 gene was found.

EGFR and KRAS Mutations in Lung Parenchyma of Subjects With EGFR/KRAS Wild-Type Lung Adenocarcinoma

The acquisition of driver mutations in non-tumoral cells appears to be very important during the carcinogenesis of adenocarcinoma (ADC). Recent studies suggest that cancer-related mutations may not necessarily be present only in malignant cells, but also in histologically “healthy cells”.Objective: to demonstrate the presence of EGFR or KRAS mutations in non-tumoral lung cells in subjects with ADC and negative mutational status.Results: mutations in EGFR or KRAS oncogenes were identified in the normal lung in 9.7% of the subjects. Exon 21 substitution L858R in EGFR was detected in two cases while the exon 19 deletion E746-A750 in the EGFR, the G12C and G12D substitutions in the KRAS were detected once. One patient presented three different mutations in the normal lung parenchyma (EGFR_L858R, KRAS_G12C and KRAS_G12D). The negative-mutation status of the tumor and the mutations detected in the “normal lung” were confirmed using highly sensitive and specific TaqMan PCR (CAST-PCR). No differences were found in terms of progression, progression-free survival or overall survival during the 18 months follow-up.Conclusions: These results confirm the presence of driver mutations in the histologically normal lung parenchyma cells in the absence of mutations coexisting with the primary tumor.

Circulating miR-618 Has Prognostic Significance in Patients with Metastatic Colon Cancer

The present study evaluated the prognostic role of circulating miRNA-618 in patients with metastatic colon cancer (mCC) and whether miR-618 gene rs2682818 single nucleotide polymorphisms (SNP) are associated with colon cancer susceptibility and expression levels of mature miR-618. In total, 104 patients with mCC before starting the chemotherapy were investigated. The expression status of circulating miR-618 in mCC was evaluated by quantitative PCR. TaqMan PCR assay was used for rs2682818 SNP genotyping. miR-618 was overexpressed in serum of mCC patients. Patients with high and intermediate expression of miR-618 had a significantly longer mean overall survival (OS) of 21 months than patients with low expression—16 months. In addition, multivariate Cox regression analysis confirmed the association between high/intermediate levels of miRNA-618 and longer OS, HR = 0.51, 95% CI: 0.30–0.86, p = 0.012. miR-618 rs2682818 SNP significantly decreased the risk of colon cancer susceptibility in both heterozygous codominant (AC vs. CC, OR = 0.39, 95% CI: 0.17–0.88, p = 0.024) and overdominant (AC vs. CC + AA, OR = 0.37, 95% CI: 0.16–0.85, p = 0.018) genetic models. Our data suggest that circulating miRNA-618 could be useful as a prognostic biomarker in mCC. Patients harboring AC rs2682818 genotype have a decreased risk for colon cancer in comparison with patients with CC and AA genotypes.