DEVELOPMENT, VALIDATION, COMPARISON AND CLINICAL IMPLEMENTATION OF DIFFERENT MULTIGENE ASSAYS FOR THE PRE-SURGICAL RISK STRATIFICATION OF INDETERMINATE THYROID FINE NEEDLE ASPIRATIONS.

Background: The aim of this PhD dissertation is to show the development, validation, comparison and preliminary clinical implementation of different multigene-based assays for the pre-surgical risk stratification of indeterminate thyroid fine-needle aspirates (FNA). In particular, this work was focused on the validation and comparison of two commercially multigene assays available for the study of the molecular alterations occurring in thyroid neoplasms, and on the development of a custom next-generation sequencing genomic panel. Methods: The two commercially available assays, one based on a real-time PCR (RT-PCR) technology and one based on a next-generation sequencing (NGS) platform were tested on a series of indeterminate thyroid FNA. Moreover, a custom NGS gene panel was designed and tested on a different series of retrospective and prospective FNA samples. Results: The commercial NGS panel showed parametric output data that were not sufficient to reliably use this panel on our clinical routine samples. Thus, the RT-PCR assay, which asses BRAF, N-H-KRAS, RET/PTC and PAX8/PPARG genomic alterations, were chosen for the subsequent clinical validation on a prospective series of n=1172 thyroid FNAs. In order to calculate the pre- and post- test risk of malignancy (ROM), only FNA with available histological follow-up (207/1006 adequate FNA, 20.6%) were included in the final study. The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was adopted for the microscopic diagnosis. FNA classified as atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) showed a 25.9% pre-test ROM whereas post-test ROM was 42.6% in mutation-positives (MT-pos) and 14.5% in mutation-negatives (MT-neg) cases, respectively. Considering the MT-positive cases, the cases harbouring BRAF-like mutations (BRAFV600E, RET/PTC1, RET/PTC3) showed a statistically significant higher ROM (80%) than those with RAS-like mutations (N-H-KRAS, PAX8/PPARγ) (32.4%, p=0.010). Follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) FNA showed a 44.1% pre-test ROM. Conversely, FN/SFN post-test ROM was 80% in MT-pos and 29.1% in MT-neg cases. Although BRAF- and RAS-like mutations were associated to different ROM even in FN/SFN cases (100% vs 71.4%), this difference did not reach a statistical significance (p=1,0). Suspicious for malignancy (SFM) FNA showed a 93% pre-test ROM; this latter figure almost overlapped to post-test ROM of both MT-pos (100%) and MT-neg (84.6%) FNAs. In particular, BRAFV600E-mutated FNAs were consistently associated with a papillary carcinoma on histology, irrespective of TBSRTC categories. Moreover, in order to switch from a RT-PCR based technology, that allows the study of alteration in only 7 genes, to a more comprehensive NGS-based multigene assay, we designed and analytically tested the performance of a custom panel. This latter, include beyond the 7-genes, additional genes which may give an additional predictive value when performed on indeterminate thyroid FNAs. Conclusions: Our preliminary data show that the 7-gene RT-PCR based test may contribute to the risk stratification in AUS/FLUS and FN/SFN categories, thanks to the significant difference in post-test ROM between MT-pos and MT-neg FNAs, confirming the high positive predictive value of BRAFV600E and BRAF-like mutations over the RAS-like genomic alterations. Moreover, the preliminary results of the custom NGS panel were satisfactory enough to expect its effective future adoption on our routine clinical samples

Phytochemical and biological study of phenolic components from Geoffroea decorticans stem bark

The stem bark of Geoffroea decorticans (Gill.ex Hook. etArn.) Burk. was used medicinally to cure several skin affections; however, phytochemical and biological antecedents were not found. Analyses of purified methanolic extract from G. decorticans bark (PFGB), realized by silylation derivatization for GC/MS, C18-CC and HPLC followed by two-dimensional TLC and UV-Vis spectroscopy, allowed to characterize nine phenolic compounds, among these, two methoxy flavonoids. Antibacterial assays of PFGB showed the highest activity (MICs = 125 μg/mL) against Staphylococcus aureus (25923) and Enterococcus faecalis (29212) ATCC strains. Moreover, PFGB showed the highest intracellular antioxidant activity at low concentration (5 μg/mL), evaluated by using the fluorescent DA-H2DCF probe on lymphocyte culture; cytotoxic effects on lymphocytes activated or not by LPS were not observed, through Trypan Blue Exclusion and MTT colorimetric assays. The results obtained from the ethnomedicinal approach of this work contribute to the scientific validation of the vulnerary medicinal use of G. decorticans.La corteza de Geoffroea decorticans (Gill.ex Hook. EtArn.) Burk. se utiliza con fines medicinales para curar diferentes afecciones de la piel; sin embargo, no encontramos antecedentes fitoquímicos y biológicos que validen las propiedades medicinales atribuidas. Analizamos el extracto metanólico purificado de corteza de G. decorticans (PFGB), por CG-EM de la muestra derivatizada por sililación, C18-CC y HPLC seguido de CCF bidimensional, y espectroscopia UV-Vis; estos métodos nos permitieron caracterizar nueve compuestos fenólicos, entre estos, dos metoxi-flavonoides. Los ensayos antibacterianos de PFGB mostraron mayor actividad (CIMs = 125 μg/mL) contra las cepas ATCC de Staphylococcus aureus (25923) y Enterococcus faecalis (29212). Además, PFGB evidenció la mayor actividad antioxidante intracelular a baja concentración (5 μg/mL), evaluada en cultivo de linfocitos, mediante el uso de sonda fluorescente DA-H2DCF; no se observaron efectos citotóxicos sobre linfocitos activados o no por LPS, a través de ensayos colorimétricos con MTT y test de exclusión con azul Tripán. Los resultados obtenidos del abordaje etnomedicinal de este trabajo, contribuyen con la validación científica del uso medicinal vulnerario de G. decorticans.Fil: Pastoriza, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Estudios Vegetales. Cátedra de Fitoquímica; ArgentinaFil: Sgariglia, Melina Araceli. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Estudios Vegetales. Cátedra de Fitoquímica; ArgentinaFil: Soberon, Jose Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Estudios Vegetales. Cátedra de Fitoquímica; ArgentinaFil: Sampietro, Diego Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Estudios Vegetales. Cátedra de Fitoquímica; Argentin

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation

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Liquid biopsy is a promising tool for genetic testing in idiopathic pulmonary fibrosis

Liquid biopsy, which allows the isolation of circulating cell-free (ccf) DNA from blood, is an emerging noninvasive tool widely used in oncology for diagnostic and prognosis purposes. Previous data have shown that serum cfDNA discriminates idiopathic pulmonary fibrosis (IPF) from other interstitial lung diseases. Our study aimed to measure plasma levels of ccfDNA in 59 consecutive therapy-naive and clinically stable IPF patients. The single nucleotide polymorphism (SNP) of the MUC5B gene promoter (rs35705950), associated with increased susceptibility of developing IPF, has been sought in plasma cfDNA and genomic DNA for comparison. Thirty-five age-and sex-matched healthy volunteers were recruited as the control group. Our results show that concentrations of small-size ccfDNA fragments were significantly higher in IPF patients than in controls and inversely correlated with lung function deterioration. Moreover, the median level of 104 ng/mL allowed discriminating patients with mild disease from those more advanced. The rs35705950 polymorphism was found in 11.8% of IPF patients and 8% of controls, with no differences. Complete concordance between ccfDNA and genomic DNA was detected in all control samples, while four out of seven IPF cases (57%) carrying the rs35705950 polymorphism were discordant from genomic DNA (7% of total IPF). Liquid biopsy is a suitable tool with optimistic expectations of application in the field of IPF. In analogy with cancer biology, finding some discrepancies between ccfDNA and genomic DNA in IPF patients suggests that the former may convey specific genetic information present in the primary site of the disease

Bacteremia and sepsis by Arcanobacterium haemolyticum in a young immunocompetent patient

We report the case of a twenty-year-old immunocompetent male patient presentingto the emergency room with pharyngitis and fever. Blood cultures were drawn and Arcanobac-terium haemolyticum (rough biotype) was recovered. The presence of the arcanolysin gene wasinvestigated at the molecular level and the upstream region was amplified and sequenced inorder to correlate it with the smooth or rough biotype. Although the isolate was susceptible topenicillin, vancomycin and gentamicin, empirical treatments first with amoxicillin/clavulanicacid (1 g/12 h) and then with ceftriaxone (1 g/12 h) failed and the infection evolved to sep-sis. Finally, treatment with vancomycin (1 g/12 h) plus piperacillin/tazobactam (4.5 g/8 h) waseffective. Lemierre?s syndrome was ruled out. To the best of our knowledge, this is the firstcase of bacteremia by A. haemolyticum reported in Argentina.Fil: Verona, Julián. Universidad de Buenos Aires; ArgentinaFil: Dirialdi, Noelia Cecilia. Universidad de Buenos Aires; ArgentinaFil: Mutti, María Virginia. Universidad de Buenos Aires; ArgentinaFil: Scioli, Marcelo Carlos. Universidad de Buenos Aires; ArgentinaFil: Thougnon Islas, Fernando Andrés. Universidad de Buenos Aires; ArgentinaFil: Curi Antún, Germán Emilio. Universidad de Buenos Aires; ArgentinaFil: Sgariglia, Lisandro Juan. Universidad de Buenos Aires; ArgentinaFil: Homar, María Guillermina. Universidad de Buenos Aires; ArgentinaFil: Legato, José Andrés. Universidad de Buenos Aires; ArgentinaFil: Costa, Agustina. Universidad de Buenos Aires; ArgentinaFil: Maldonado, María Laura. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Universidad de Buenos Aires; ArgentinaFil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Litterio Bürki, Mirta R.. Universidad de Buenos Aires; Argentin

RNA-Based Assay for Next-Generation Sequencing of Clinically Relevant Gene Fusions in Non-Small Cell Lung Cancer

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC

Use of hydroxychloroquine in hospitalised COVID-19 patients is associated with reduced mortality: Findings from the observational multicentre Italian CORIST study

Background: Hydroxychloroquine (HCQ) was proposed as potential treatment for COVID-19. Objective: We set-up a multicenter Italian collaboration to investigate the relationship between HCQ therapy and COVID-19 in-hospital mortality. Methods: In a retrospective observational study, 3,451 unselected patients hospitalized in 33 clinical centers in Italy, from February 19, 2020 to May 23, 2020, with laboratory-confirmed SARS-CoV-2 infection, were analyzed. The primary end-point in a time-to event analysis was in-hospital death, comparing patients who received HCQ with patients who did not. We used multivariable Cox proportional-hazards regression models with inverse probability for treatment weighting by propensity scores, with the addition of subgroup analyses. Results: Out of 3,451 COVID-19 patients, 76.3% received HCQ. Death rates (per 1,000 person-days) for patients receiving or not HCQ were 8.9 and 15.7, respectively. After adjustment for propensity scores, we found 30% lower risk of death in patients receiving HCQ (HR=0.70; 95%CI: 0.59 to 0.84; E-value=1.67). Secondary analyses yielded similar results. The inverse association of HCQ with inpatient mortality was particularly evident in patients having elevated C-reactive protein at entry. Conclusions: HCQ use was associated with a 30% lower risk of death in COVID-19 hospitalized patients. Within the limits of an observational study and awaiting results from randomized controlled trials, these data do not discourage the use of HCQ in inpatients with COVID-19