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Abstract Background Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC. Methods A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands. Results The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 ± 686) than in normal adrenal glands (362 ± 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 ± 196) was significantly lower than in normal adrenal glands (3614 ± 949; p = 0.01). Conclusion On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

Background: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC.Methods: A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands.Results: The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 +/- 686) than in normal adrenal glands (362 +/- 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 +/- 196) was significantly lower than in normal adrenal glands (3614 +/- 949; p = 0.01).Conclusion: On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC. [ABSTRACT FROM AUTHOR]

Circulating tumor cells and cell-free nucleic acids are novel diagnostic, prognostic and predictive tools for non-invasive and cost-effective cancer detection in liquid biopsy. Carbonic anhydrase IX (CAIX) has been proposed as a biomarker in urogenital tumors and urine sediment. Our aim was to evaluate CAIX full-length percentage (CAIX FL%) in urine-cell-free RNA (cfRNA) and its relationship with tumor-cell-associated RNA (TC-RNA). CAIX FL% was quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in patients with prostate, kidney or bladder carcinoma. When cfRNA and TC-RNA were analyzed, CAIX FL% was significantly higher in urine samples from cancer patients than from controls. Using a 10% cutoff for CAIX FL%, specificity, sensitivity, positive and negative predictive values, as well as accuracy for TC-RNA were higher than for cfRNA in all urogenital cancers, but varied according to tumor type. CAIX FL% distribution in TC-RNA differed significantly ( p < 0.001) between control and tumor samples (37.5% and 96.2%, respectively); similar results were obtained for each tumor type. Additionally, the 10% cutoff showed a 77.9% concordance between TC-RNA and cfRNA. In conclusion, urine is proposed as an alternative biofluid for investigating CAIX FL% in urogenital cancers, and this parameter can be reliably measured as cfRNA and TC-RNA with different predictive capabilities depending on tumor type.

Detection of SARS-CoV-2 in bronchoalveolar lavage (BAL) is considered as a promising alternative method to detect COVID-19 infection. STANDARD™ M10 SARS-CoV-2 assay on 150 negative and 50 positives BAL samples for SARS-CoV-2 showed 96 % sensitivity, 100 % specificity compared to Allplex™ SARS-CoV-2 assay and a 31.25 genomic copies/mL limit of detection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Outside of the submitted work, AA and GMR received honoraria from SD Biosensor for presentations at meetings within educational workshops., (Copyright © 2024. Published by Elsevier Inc.)

Immunocompromised patients still experience unpredictable courses of COVID-19, despite that effective vaccines and drugs against SARS-CoV-2 are now available. Antiviral combination regimens may have a role in SARS-CoV-2 infection in immunocompromised hosts, but current knowledge is still limited. We describe the case of a 73-year-old Italian man affected by follicular lymphoma with persistent SARS-CoV-2 infection who was successfully treated with co-administration of oral antivirals (10-day molnupiravir and nirmatrelvir/ritonavir). The therapy was well tolerated both from a clinical and biochemical standpoint, with no signs of toxicity. We also performed a scoping review, to sum up available knowledge on combined antiviral regimens including remdesivir, molnupiravir, or nirmatrelvir/ritonavir. Pending further studies on larger cohorts of patients, our report is consistent with available pre-clinical and clinical data, supporting the possible use of combination therapy in selected difficult-to-treat COVID-19 cases., Competing Interests: Declarations of competing interest The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We present a brief commentary illustrating the current COVID-19 outpatient treatment options in Italy. We also report our experience setting up a service dedicated to these patients in the wake of the rise in COVID-19 cases observed in January 2022. We also gathered data on the daily costs faced by our outpatient service, based at a tertiary care center located in Florence, Italy. We present them with some considerations on future outlooks on the use of outpatient treatment in COVID-19.

Although accumulating data have investigated the effect of SARS-CoV-2 mutations on antibody neutralizing activity, less is known about T cell immunity. In this work, we found that the ancestral (Wuhan strain) Spike protein can efficaciously reactivate CD4+ T cell memory in subjects with previous Alpha variant infection. This finding has practical implications, as in many countries only one vaccine dose is currently administered to individuals with previous COVID-19, independently of which SARS-CoV-2 variant was responsible of the infection. We also found that only a minority of Spike-specific CD4+ T cells targets regions mutated in Alpha, Beta and Delta variants, both after natural infection and vaccination. Finally, we found that the vast majority of Spike-specific CD4+ T cell memory response induced by natural infection or mRNA vaccination is conserved also against Omicron variant. This is of importance, as this newly emerged strain is responsible for a sudden rise in COVID-19 cases worldwide due to its increased transmissibility and ability to evade antibody neutralization. Collectively, these observations suggest that most of the memory CD4+ T cell response is conserved against SARS-CoV-2 variants of concern, providing an efficacious line of defense that can protect from the development of severe forms of COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mazzoni, Vanni, Spinicci, Capone, Lamacchia, Salvati, Coppi, Antonelli, Carnasciali, Farahvachi, Giovacchini, Aiezza, Malentacchi, Zammarchi, Liotta, Rossolini, Bartoloni, Cosmi, Maggi and Annunziato.)

Background: The emergence of SARS-CoV-2 variants of concern (VOCs) for increased transmissibility and being potentially capable of immune-escape mandates for epidemiological surveillance. Genomic alterations present in VOCs can affect the results of RT-qPCR assays for routine diagnostic purposes, leading to peculiar profiles that can be used for rapid screening of variants. This study reports a peculiar profile observed with the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay and VOC-Alpha (202012/01, lineage B.1.1.7, also named VOC-UK), which was the first identified SARS-CoV-2 VOC., Methods: Samples were analyzed by two RT-qPCR assays: the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (ASFR, Seegene Technologies Inc; Seoul, South Korea) and the TaqPath COVID-19 RT-PCR (Thermo Fisher Scientific, USA). Definition of the SARS-CoV-2 variant was carried out by Sanger sequencing of the relevant S-gene regions and, in some cases, by whole genome sequencing (WGS) using the ARTIC-nCoV workflow on a MiniION (Oxford Nanopore Technologies, Oxford, UK) or a Illumina MiSeq platform (San Diego, California, USA)., Results: Of the 173 SARS-CoV-2-positive specimens, all those of lineage B.1.1.7 (N=71) showed an average Cq difference between the N and S genes of +11±2 (range, +8/+15). None of the other specimens, including several different lineages (Wild-type for the analyzed regions, N=22; Gamma, N=63; Delta, N=9; B.1.258Δ, N=3; B.1.160, N=3; B.1.177.7, N=1; B.1.1.420, N=1), exhibited a similar difference in Cq values., Conclusions: The peculiar pattern of delayed N gene positivity could constitute a convenient method for VOC-Alpha screening, simultaneous to viral detection, when using the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay., Competing Interests: DECLARATION OF COMPETING INTEREST Dr. Antonelli reports personal fees from Accelerate diagnostics, personal fees from Arrow diagnostics, personal fees from Menarini, personal fees from Seegene, non-financial support from SymCel, outside the submitted work. Dr. Rossolini reports grants, personal fees and non-financial support from Accelerate Diagnostics, personal fees from Becton Dickinson, grants and personal fees from bioMérieux, grants and personal fees from Cepheid, grants and personal fees from Elitech, grants and personal fees from Merck, grants and personal fees from Nordic Pharma, personal fees from Pfizer, grants from Seegene, grants and personal fees from Shionogi, personal fees and other from Venatorx, grants and personal fees from Zambon, personal fees from Roche, personal fees from Thermo Fisher, personal fees and non-financial support from Beckman Coulter, grants, personal fees and non-financial support from Menarini, grants from Arrow, grants from Symcel, personal fees from QPex, grants from DID, grants from Hain Lifescience GmbH, grants from GenePoc, grants from SetLance, grants and personal fees from Angelini, grants from Qvella, grants from Qlinea, personal fees from Qiagen, grants from Biomedical Service, grants from Liofilchem, outside the submitted work. Dr. Baccani reports for Diesse-Diagnostica Senese, outside the submitted work. All other authors have nothing to disclose., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A., Design: Case-control laboratory study., Setting: University institute and university hospital., Patient(s): Patients with uterine fibroids (n = 26)., Interventions(s): Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy., Main Outcome Measure(s): Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines., Result(s): The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P 2 , S1P 3 , and S1P 5 receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P 2 . The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P 2/3 were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells., Conclusion(s): This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Malignant melanoma is an aggressive form of skin cancer, which develops from the genetic mutations of melanocytes - the most frequent involving BRAF and NRAS genes. The choice and the effectiveness of the therapeutic approach depend on tumour mutation; therefore, its assessment is of paramount importance. Current methods for mutation analysis are destructive and take a long time; instead, Raman spectroscopy could provide a fast, label-free and non-destructive alternative. In this study, confocal Raman microscopy has been used for examining three in vitro melanoma cell lines, harbouring different molecular profiles and, in particular, specific BRAF and NRAS driver mutations. The molecular information obtained from Raman spectra has served for developing two alternative classification algorithms based on linear discriminant analysis and artificial neural network. Both methods provide high accuracy (≥90%) in discriminating all cell types, suggesting that Raman spectroscopy may be an effective tool for detecting molecular differences between melanoma mutations., (© 2020 Wiley-VCH GmbH.)

Objective: To study the molecular mechanisms involved in the appearance of the fibrotic trait in endometriosis by investigating whether the signaling pathway of the bioactive sphingolipid sphingosine 1-phosphate (S1P) was altered in endometriotic lesions., Design: Case-control laboratory study., Setting: University research institute and university hospital., Patient(s): A total of 75 women, with and without endometriosis, were included in the study., Interventions(s): Endometrial samples were obtained from women affected (n = 15 endometrioma [OMA]; n = 30 deep infiltrating endometriosis [DIE]) and not (n = 30) by endometriosis by means of laparoscopic surgery, followed by clinical and imaging investigation and checking for the expression of fibrosis markers and genes implicated in S1P metabolism and signaling by means of real-time polymerase chain reaction., Main Outcome Measure(s): The role of the S1P signaling axis in endometriosis-associated fibrosis was studied in vitro, where RNA interference approaches were used to investigate if S1P synthesis by sphingosine kinases (SKs) and specific S1P receptors (S1PRs) are implicated in the profibrotic effect of the cytokine transforming growth factor (TGF) β1., Result(s): mRNA expression analysis of S1PR demonstrated a deep dysregulation of S1P signaling in endometriosis, characterized by increased expression of fibrosis markers: S1P 1 was transcriptionally more expressed in OMA, and S1P 3 and S1P 5 mRNA levels were significantly augmented in both OMA and DIE. SK1 and its activating protein calcium- and integrin-binding protein 1 (CIB1) were significantly up-regulated in OMA and DIE. A crucial role for the SK/S1PR axis in the profibrotic effect elicited by TGFβ1 was highlighted in vitro., Conclusion(s): The S1P signaling axis may represent a useful biomarker or innovative pharmacologic target for endometriosis., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: In cervical cancer screening programs, women with abnormal cytology and confirmation by biopsy are referred for colposcopy for histological evaluation., Methods: We characterized the presence and the genotype of HPV by Linear Array HPV genotyping assay in cytological samples collected from about 400 women undergoing conization, with reported high CIN grade after biopsy., Results: The most prevalent genotype was HPV 16, with an increasing presence depending on the severity of the CIN and with the highest incidence in the 26-35 age range. In the group of younger women (<25) we found the highest percentage of CIN3 (39.3%) and the lowest of CIN1 (17.9%). An increase of CIN1 with increasing age was observed. A different distribution of HPV presence was observed depending on CIN grade (P<0.001): CIN1 HPV negative samples were 46.3%, CIN2: 5.8% and CIN3: 1.4%. Interesting, in the analyzed cohort, we observed the presence of 30% of CIN1. Moreover, within CIN1, 85% of them were associated to negative HPV detection, this observation suggested that the detection of HPV presence may be useful to identify low CIN grade that should be reconsidered for surgical treatment., Conclusions: These findings suggest implementing the protocol for the management of women with high risk precancer lesions, with a further HPV test before surgical treatment. The evaluation of HPV presence and genotype before conization might represent a useful tool in reducing or postpone the conization treatment.

Endometriosis is an estrogen-dependent inflammatory gynecological disease. Increased estrogen activity and progesterone resistance are the main hormonal substrate of this disease and are associated with inflammatory response and debilitating symptoms, including pain and infertility. Estrogens and progesterone act via their specific nuclear receptors. The regulation of receptor expression by epigenetics maybe a critical factor for endometriosis. The present review aims to discuss the epigenetic mechanisms related to the expression of estrogen receptors (ERs) and progesterone receptors (PRs) in patients with endometriosis, including two classic epigenetic mechanisms: DNA methylation and histone modification, and, other non-classic mechanisms: miRNAs and lncRNA. Several in vitro and in vivo studies support the key role of epigenetics in the regulation of the expression of ERs and PRs, which may provide new molecules and targets for the diagnosis and treatment of endometriosis.

Endometrial cancer is the commonest gynecological cancer, the majority is endometrioid type, diagnosed at an early stage with 69-88% 5-year survival. Low-grade endometrial cancers have low recurrence rates and often do not receive adjuvant therapy; however, a subset of these patients will have poor outcomes and would benefit from adjuvant treatment has been challenging. We evaluate the circulating cell-free DNA (ccfDNA) in a patient with low-risk endometrial cancer in order to identify the presence of molecular markers associated with risk of recurrence. The evaluation of mutation profile was performed by next-generation sequencing (NGS) in primary tumor formalin-fixed paraffin-embedded (FFPE) tissue and in circulating tumor DNA (ctDNA). We identified a specific mutational profile in ctDNA, different from primary tumor tissue suggesting that the clone involved in the relapse may be different in comparison to the most represented in the primary tumor. These findings open new prospective and new wonderings. The molecular characterization of tissue may be useful for setting new target personalized therapy even in the treatment of endometrial cancer, moreover, endometrial cancer at low risk should be not underestimated for the incidence of relapse, and for this evaluation the molecular characterization may be useful. Moreover, these results suggest that the single analysis of primary tumors may be not sufficient for setting a specific personalized therapy targeted to avoid the relapse but may be necessary to join the molecular characterization of liquid biopsy to primary tissue.

Uterine carcinosarcomas are biphasic neoplasms consisting of mixed epithelial and mesenchymal elements, representing less than 5% of all uterine malignancies. Carcinosarcomas are rare, although the most common cause of uterine cancer-specific death. Few information is available on the pathogenesis, and molecular characterization is poorly investigated. Consequently, the treatment has not changed over the last years and is far too being tailored, consisting of surgery and traditional chemotherapy and radiotherapy. Molecular characterization of liquid biopsy by circulating tumor DNA (ctDNA)/circulating cell-free DNA (ccfDNA) evaluation in a patient with uterine carcinosarcoma. Here, we describe a case report of an 83-year-old woman with carcinosarcomas, stage T3aN0M0. Cancer cells did not express estrogen nor progesterone receptors, while p53 and p16 were positive. Molecular characterization of ccfDNA and of ctDNA was performed by quantitative PCR, amplification-refractory mutation system technology. The presence of phosphatidylInositol-4,5-bisphosphate 3-Kinase catalytic subunit alpha p.E545A mutation was detected in plasma. This approach may suggest the use of liquid biopsy and the development of specific targeted therapy for precision personalized medicine even in rare carcinosarcomas.

Background: Detection and genotyping of human papillomavirus (HPV) has gained increasing importance in cervical cancer prevention and treatment of cervical intraepithelial neoplasia (CIN). This study aims to determine the HPV type distribution in cervical specimens obtained from women diagnosed with CIN. We evaluated in a selected Italian population the distribution of HPV genotypes., Methods: Cervical samples were collected from women undergoing laser CO2 conization for high grade at Colposcopic Laser Surgery Unit of the Careggi University Hospital and at the Colposcopy Service of Local Health Unit Toscana Centro in Florence, Italy, between September 2014 and February 2017. HPV genotyping was performed using the LINEAR ARRAY® HPV Genotyping Test., Results: Three hundred and six patients were enrolled. HPV infection was detected on 244 samples (79.7%). A different rate of mono- and poly-infections was observed, with higher poly-infection rates in younger women. Moreover, depending on different age groups (clustered in 5-years interval from 22 to 69 years old) significant different distribution of HPV was fund as genotype, phylogenetic type and cancer-related risk., Conclusions: Our results suggest that some physiological conditions (i.e. menopause), could influence selection and clearance of specific HPV genotypes. The results of this study represent the basis for supporting the HPV genotyping as clinical tool providing benefits in the management of women with high CIN grade.

The incidence of endometrial cancer (EC) is increasing in developed countries. The most frequent is the endometrioid subtype with usually good prognosis; nevertheless, some cases escape this paradigm and may have recurrence. A recent study from The Cancer Genome Atlas suggested to implement the EC analysis by molecular profile for improving diagnosis, prognosis, and therapeutic treatment. The present preliminary study was performed on 15 G3 endometrioid endometrial cancers (G3 EEC) for the identification of somatic mutations in a panel of specific exons in selected genes as ARID1A, CTNNB1, KRAS, PIK3CA, POLE, PTEN, and TP53. The combined procedure, based on the Sanger sequencing and PCR-high-resolution melting analysis, allowed the identification of variations of the selected gene panel in most of patients (93%) of our cohort. The overall evaluation of mutational load exhibited that the most frequent mutated genes were PTEN (93%), followed by PIK3CA (47%) suggesting a deep involvement of PI3K pathway alteration in G3 EEC. Mutations in TP53 (27%), ARID1A (27%), POLE (13%), and at the lower level in KRAS and CTNNB1 (7%) were also observed (exclusively in FIGO III stage patients). The evaluation of the mutations of our proposed panel (ARID1A, CTNNB1, KRAS, PIK3CA, POLE, PTEN, TP53) is suitable to improve the characterization of G3 EEC and could suggest targetable pathways for development of personalized treatments.

Endometrial cancer (EC) comprises a biological and clinical heterogeneous group of tumors. Several genetic alterations are involved in the development and progression of EC, and may be used for targeted therapy, particularly in patients with advanced‑stage EC. In the present study, a combined procedure was developed based on polymerase chain reaction (PCR)‑high resolution melting analysis (HRMA) and Sanger sequencing for the evaluation of somatic mutations in selected phosphoinositide 3‑kinase (PI3K) catalytic subunit α (PIK3CA; exons 1, 9 and 21) and phosphatase and tensin homolog (PTEN; exons 5, 6, 7 and 8) exons. This combined procedure has the specificity and sensitivity of the two techniques, and overcomes their limitations. A pilot study was performed on 18 selected homogenous EC samples, of grade 3 endometrioid subtype (G3 EEC). First, the feasibility of the combined procedure was investigated to properly identify the presence of somatic mutations on PIK3CA and PTEN, the variations identified were analyzed using Catalogue of Somatic Mutations in Cancer, PolyPhen‑2 and Mutation Taster software, and the frequency of mutations/variations was determined in the selected samples. The evaluation of mutational load revealed that the majority of the G3 EEC samples exhibited PIK3CA mutations (39%) and PTEN mutations (67%), and the majority of the samples (83%) had mutations in at least one of the two genes, and 33% had mutations in the two genes. The results of the present pilot study suggested that the cost‑effective combined PCR‑HRMA and Sanger sequencing procedure may be suitable for identification of PTEN and PIK3CA mutations in G3 EEC and that their frequency was consistent in G3 EEC, indicating that the PI3K pathway serves a pivotal function that may have potential for defining targeted therapy for the treatment of G3 EEC.

The aim of this study was to develop a scoring system of the immunohistochemical (IHC) expression of luteinizing hormone/human chorionic gonadotropin receptor (LHCG-R) in endometrial cancer (EC) patients. Nonconsecutive hysterectomy specimens containing EC collected from April 2013 to October 2015 were selected. Hematoxylin-eosin stained sections from each case were reviewed and representative sections from each tumor were selected. IHC staining was performed for the detection of LHCG-R. The percentage of stained cells and the staining intensity were assessed in order to develop an immunohistochemical score. Moreover, we examined the correlation of the score with grading and lymphovascular space invasion (LVSI). There was a statistically significant positive correlation between grading and IHC scoring ( p = 0.01) and a statistically significant positive correlation between LVSI and IHC score ( p < 0.01). In conclusion, we suggest that the immunohistochemical score presented here could be used as a marker of bad prognosis of EC patients. Nevertheless, further studies are needed in order to validate it. The study was registered in the Careggi Hospital public trials registry with the following number: 2013/0011391.

Background: The Bombay phenotype is a rare genetic trait which is characterized by the absence of A, B and H antigens on red cells as well as in body secretions. The serum shows the presence of antibodies against antigen H. Patients with this rare blood type are not easily transfusable. We had observed a woman aged 18, at the 20th week of pregnancy, native of Sri Lanka, with an IgG and IgM class anti-H. We report the case and the clinical issues arisen., Materials and Methods: The determination of ABO, Rh[D] group, the indirect antiglobulin test (IAT) were performed in tube techniques and in neutral gel microcolumn. Detection for antibodies was performed using ID-Card LISS-Coombs microtubes, in solid phase and with tube techniques. For molecular analysis, the FUT1 and FUT2 genes were sequenced using BigDye terminator v1.1. The study of FUT2 gene was performed after extraction of mRNA using Qiagen kit RNase and then reverse-transcribed into cDNA., Results: The Bombay phenotype was confirmed by serological and molecular analysis techniques. The patient, in collaboration with a cultural mediator, was informed of her immunohaematological condition and a program of assistance was proposed to her. Unfortunately the patient did not return for the next visit, despite of a telephone reminder. During childbirth a haemorrhage occurred and a request of compatible blood for an urgent transfusion arrived at our transfusion service. Fortunately, the haemorrhage was arrested and the patient didn't need to have any transfusions., Conclusion: This case emphasizes the need for an efficient management of rare blood types that are more and more frequent as a result of migration. It is necessary to organize, in strategic points of the national territory, reference centres with better diagnostic capabilities and implement freezing of red blood cells with rare phenotype for diagnostic and therapeutical use. Communication issues are as well important in dealing with this emerging phenomenon.

Background: To identify molecular biomarkers for tumor diagnosis and monitoring of disease progression, several noninvasive tests on liquid biopsy have been proposed for different cancers including those of urogenital origin. Among biomarkers, carbonic anhydrase IX (CAIX) has gained attention as it regulates extracellular pH and induces cytoplasmic alkalization contributing to malignant progression and poor treatment outcome. Works on tissues suggested the potential use of CAIX as a tumor biomarker for urogenital malignancies, but only few studies have been performed on its detection in urine., Scope: The aim of the present study is the measurement of CAIX messenger RNA (mRNA) in urine sediments of patients affected by kidney, prostate, and bladder cancers to evaluate the clinical sensitivity and specificity of the test., Procedures: The quantification of the total CAIX mRNA concentration and of its full-length isoform (CAIX FL) have been performed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) on RNA extracted from urine sediments of patients affected by urogenital cancers., Results: Urinary total CAIX mRNA expression resulted to be lower in patients with kidney and prostate cancer in comparison with the control group, but no statistically significant difference could be evidenced for bladder cancer. The evaluation of the relative percentage of FL isoform mRNA (FL%) showed a significant increase of FL% in urine from patients with cancer (median = 70.8%) in comparison with the healthy subjects (median = 2.6%) and this finding was confirmed for each cancer type separately. The comparison among receiver operating characteristic curves for total CAIX mRNA, CAIX FL mRNA, and FL% indicated that FL% shows the best diagnostic performance with 90% sensitivity and 72% specificity. Comparison of the results obtained in urine with those found in the corresponding tissues indicated 80% concordance., Conclusions: The CAIX mRNA expression in urine sediments can be considered a surrogate marker of CAIX expression in tumor tissues of urogenital origin. In particular, the analysis of FL% possesses the best characteristics to be a suitable noninvasive biomarker for urogenital cancer diagnosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Inappropriate handling of blood samples might induce or repress gene expression and/or lead to RNA degradation affecting downstream analysis. In particular, sample transport is a critical step for biobanking or multicenter studies because of uncontrolled variables (i.e., unstable temperature). We report the results of a pilot study implemented within the EC funded SPIDIA project, aimed to investigate the role of transport and storage of blood samples containing and not containing an RNA stabilizer., Methods: Blood was collected from a single donor both in EDTA and in PAXgene Blood RNA tubes. Half of the samples were sent to a second laboratory both at room temperature and at 4°C, whereas the remaining samples were stored at room temperature and at 4°C. Gene expression of selected genes (c-FOS, IL-1β, IL-8, and GAPDH) known to be induced or repressed by ex vivo blood handling and of blood-mRNA quality biomarkers identified and validated within the SPIDIA project, which allow for monitoring changes in unstabilized blood samples after collection and during transport and storage, were analyzed by RT-qPCR., Results: If the shipment of blood in tubes not containing RNA stabilizer is not performed under a stable condition, gene profile studies can be affected by the effects of transport. Moreover, also controlled temperature shipment (4°C) can influence the expression of specific genes if blood is collected in tubes not containing a stabilizer., Conclusion: The use of dedicated biomarkers or time course experiments should be performed in order to verify potential bias on gene expression analysis due to sample shipment and storage conditions. Alternatively, the use of RNA stabilizer containing tubes can represent a reliable option to avoid ex vivo RNA changes.

Background: In order to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time., Methods: SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm., Results/conclusions: 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on pre-analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent pre-analytical conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses - [1]; Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience - [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience - [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality - [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis. Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

Background: Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies., Methods: Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR)., Results: The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile., Conclusions: The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia., (© 2015 Wiley Periodicals, Inc.)

In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach., (Copyright © 2015 Elsevier Inc. All rights reserved.)

One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

Studies on miRNA profiling revealed that a large number of them are significantly deregulated in human cancers. The molecular mechanisms of this deregulation are not totally clarified, even if genetics and epigenetics are frequently involved. Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in the human genome. A SNP into miRNA gene might affect the transcription of primary miRNA, its processing and miRNA-mRNA interaction. We investigated the distribution of sequence variants of miR-146a, miR-196a2, miR-499 and miR-149 in colorectal cancer (CRC) and their effect on miRNA expression. Each variant was identified with HRM. For miR-499 we demonstrated a significant reduction of its expression in CRC connected to a specific genotype. To evaluate the epigenetic effects on miRNA genes in CRC, we investigated the influence of DNA methylation on miR-34b, miR-34c and miR-9-1 expression. We aimed to verify the relationship between the methylation status of these miRNA genes and their relative expression in tumor samples. For the quantification of DNA methylation we adopted a method based on Differential High Resolution Melting (D-HRM)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: In human cancers, carbonic anhydrase IX (CAIX) influences cell proliferation and tumor progression, maintaining intracellular and extracellular pH under hypoxic conditions. An alternative CAIX isoform, lacking of exons 8-9 (AS) and independent from the levels of hypoxia, was recently demonstrated in cancer cells. AS-CAIX competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study, we evaluated mRNA expression of the 2 CAIX isoforms and their clinical relevance in bladder cancers and urine sediments., Materials and Methods: We measured mRNA expression of FL- and AS-CAIX isoforms in tumor tissues and benign mucosa from 45 patients with bladder transitional cell carcinoma. The expression of the 2 isoforms was also measured in urine sediment of 81 bladder cancer patients and 93 control subjects., Results: Expression of FL-CAIX mRNA was lower than AS-CAIX in benign mucosa (P = 0.006) whereas in paired bladder cancers FL-CAIX mRNA was higher (P = 0.007). Consequently, the percentage of FL-CAIX in bladder cancers [median: 62.6%] was significantly higher than in benign mucosa [15.0%] (P < 0.0001). In the urinary sediments of bladder cancer patients FL-CAIX mRNA was significantly higher in comparison with normal controls (P = 0.003). FL-CAIX percentage appeared dramatically higher in urine sediments of bladder cancer patients [64.5%] in comparison with controls [7.5%] (P < 0.0001). In addition, FL-CAIX% was significantly different in sediments from pTa-pT1 and ≥ pT2 patients [51.5% and 91.7%, respectively] (P = 0.016). Stratification according tumor grade indicated that FL-CAIX% was significantly lower in G1 bladder cancers [33.3%] in comparison with G2-G3 [88.6%] (P = 0.005) The clinical sensitivity for FL-CAIX% in urine sediments was 0.93, with a 0.76 specificity. Using the same cut-off positive predictive value (PPV) was 0.78, whereas negative predictive value (NPV) was 0.93., Conclusions: Our results seem to indicate that in bladder cancers and related urine sediments, FL-CAIX is the prevalent and is the most accurate clinically relevant variant surrogate of hypoxic stress., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Although important improvements of downstream molecular in vitro diagnostics assays based on RNA from blood were made, the pre-analytical workflow is still poorly defined., Methods: We performed a multicenter study within the EU-granted SPIDIA project to investigate blood collection and shipping influence on the following RNA quality parameters: yield, purity, integrity, RT-qPCR interference and IL1B, IL8, FOS and GAPDH gene expression. Two models were designed: Exp A. Ten laboratories collected blood from an own donor into two different tubes (with or without stabilizer) and extracted RNA at two different times; Exp B. Blood was drawn from a single donor and shipped to ten laboratories in two different tubes (with or without stabilizer) for RNA extraction., Results: In both models and collection tubes, reliable results were obtained for purity, yield, GAPDH expression, and interferences. A substantial variation in RIN (Exp A) and in transcription levels of IL1B, IL8 and FOS (Exp B) was observed for blood collected in tube without stabilizer tubes. Overall the variability was higher among data obtained from unstabilized blood samples., Conclusions: We defined the experimental setup for a larger ring trial throughout Europe. The chosen downstream analyses verified their potential, serving as adequate markers to test the quality of blood RNA., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The presence of sequence variants in miRNA genes may influence their processing, expression and binding to target mRNAs. Since single miRNA can have a large number of potential mRNA targets, even minor variations in its expression can have influences on hundreds of putative mRNAs., Methods: Here, we evaluated 101 paired samples (cancer and normal tissues) from non-small cell lung carcinoma (NSCLC) patients to study the genotype distribution of single nucleotide polymorphisms (SNPs) in miR-146a (rs2910164 C-G), miR-149 (rs2292832 C-T), miR-196a2 (rs11614913 C-T) and miR-499 (rs3746444 G-A) and their influence on the expression of respective miRNAs., Results: Relative expression of miR-146a, miR-149 and miR-499 were comparable in NSCLC and in paired control tissues. On the contrary, we clearly detected a significant increase (p<0.001) of miR-196a2 expression in NSCLC. In particular we found a significant association between miR-196a2 CC genotype and high expression, whereas TT geno-type showed a very low expression in comparison to both CT (p<0.005) and CC patients (p<0.01). We did not find any association between miR-149, miR-196a2 and miR-499 genotype and risk of NSCLC. Conversely, CG genotype of miR-146a appeared associated to an increased risk for NSCLC (p=0.042 and 1.77 OR)., Conclusions: Our results seem to demonstrate that sequence variants of miR-196a2 can have an influence on its expression, while miR-146a can have a role in increasing the risk of NSCLC.