CORRELAZIONE TRA RISPOSTA RADIOLOGICA PRECOCE E VARIAZIONI DI ALFA-FETOPROTEINA IN PAZIENTI CON EPATOCARCINOMA TRATTATI CON SORAFENIB

Riassunto Scopo della tesi Valutare retrospettivamente la correlazione tra risposta radiologica precoce, variazioni di alfafetoproteina (AFP) e sopravvivenza in pazienti cirrotici in classe A di Child-Pugh con epatocarcinoma (HCC) trattati con Sorafenib. Materiali e metodi Sono stati raccolti i dati clinici relativi a pazienti con HCC e cirrosi in classe A di Child-Pugh trattati con Sorafenib (400mg due volte al giorno) dal 2008 al 2012. Sono stati inclusi nello studio solo pazienti con misurazioni di AFP ed esami TC eseguiti presso la nostra struttura prima del trattamento e a 3 mesi dall’inizio del trattamento. Gli esami TC sono stati riesaminati per valutare la risposta radiologica secondo i criteri RECIST 1.1, mRECIST ed EASL. È stato inoltre calcolato il volume della lesione captante il mezzo di contrasto in fase arteriosa mediante software di segmentazione semi-automatica. Infine, è stata calcolata la densità nelle acquisizioni in fase arteriosa della lesione di maggiori dimensioni. È stata eseguita un’analisi statistica per analizzare la correlazione tra la sopravvivenza dei pazienti e i parametri raccolti, con particolare riferimento alla risposta radiologica precoce e alle variazioni di AFP. Risultati Lo studio ha incluso 22 pazienti (18 maschi, età media 68 anni), di cui quattro (18.2%) in classe A del BCLC, 12 (54.5%) in classe B e 6 (27.3%) in classe C. la durata media del trattamento è stata di 10 mesi ed il follow-up medio di 14 mesi. È stata registrata una buona riproducibilità intra-osservatore nella valutazione della risposta radiologica (k=0,83-0,86) ed una buona concordanza tra mRECIST ed EASL (k=0,86) a fronte di una bassa concordanza tra questi criteri e i RECIST 1.1 (k=0,48- 0,50). In un paziente il volume tumorale non è stato calcolabile per impegno esteso miliariforme. La variazione media del volume tumorale a 3 mesi è stata di 1,76±2,17, con incremento volumetrico in 12/21 pazienti (57,1%). Al termine dello studio sono stati registrati undici decessi, con sopravvivenza cumulativa a 12 mesi di 65,9%. All’analisi statistica non è stata osservata alcuna correlazione tra sopravvivenza e variazioni di AFP o risposta radiologica secondo i diversi criteri proposti. Di contro la sopravvivenza è risultata significativamente correlata alla durata del trattamento con Sorafenib ed alle variazioni del volume tumorale. In particolare, la sopravvivenza cumulativa a 12 mesi è stata del 90% in pazienti con volume ratio ≤1,17 e del 40% in pazienti con volume ratio >1,17 (p=0,04). Conclusioni: Pur con una buona riproducibilità, i criteri radiologici di risposta tumorale non sembrano avere un valore prognostico in termini di sopravvivenza in pazienti con HCC trattati con Sorafenib, non essendo in grado di stratificare adeguatamente i pazienti. Al contrario, il calcolo della variazione volumetrica della lesione, pur richiedendo un tempo di ricostruzione di alcuni minuti, consente di identificare precocemente i pazienti con adeguata risposta al trattamento; in particolare un incremento volumetrico a 3 mesi >1,17 rappresenta un fattore prognostico sfavorevole

Do we really need contrast medium? Quantitative measurement of ADC for disease activity assessment in paediatric Inflammatory Bowel Disease

Objective To establish an ADC cut-off value to classify active and non-active lesions in inflammatory bowel disease (IBD). Methods and Materials We reviewed 167 paediatric Magnetic resonance enterographies (MREs) executed for suspected IBD. Data such as bowel wall thickness, DWI restriction, stenosis and pre-stenotic dilatation, comb sign, fat stranding, reactive lymph nodes, and contrast enhancement pattern were collected. In the ADC map, quantitative assessment of disease activity was measured (max 3 lesions/exam) placing 3 regions of interest (ROIs) in the areas of highest inflammation (based on qualitative analysis of the DWI sequence) and the mean ADC value was calculated. Similarly, we assessed ADC values of the bowel wall in those patients were no IBD lesions were found, placing the ROIs at a standardised site. Ileocolonoscopy, esophagogastroduodenoscopy or surgery with histology and video-capsule endoscopy were used as the standards of reference. Results Due to rigid inclusion criteria only 34 patients were eventually enrolled in the study. Radiological findings of IBD were identified in 29 MREs and 44 lesions were detected, using two different MRI scanners (1.5- and 3-tesla). The remaining 6 patients had negative MRE. A total of 56 bowel segments were included in the study. Acute disease was found in 39 lesions (69.6%), whereas 17 (30.4%) were classified as non-active lesions or healthy segments. Active ADC values were lower compared to the ones of non-active portions. For each scanner an ADC cut-off value to discern active and non-active segments was found. Sensitivity was 0.91 for both the MREs, and specificity was 0.89 and 0.81 for 1.5T and 3T respectively. Inter-rater agreement on disease activity between ADC values and MRE results and between ADC values and the standard of reference were very good for both 1.5T and 3T. Conclusions DWI is a powerful tool to detect IBD involvement of a bowel segment and ADC map can provide a scanner-based quantitative measurement of disease activity to separate active and non-active lesions

Quantitative Analysis of Apparent Diffusion Coefficient for Disease Assessment in Paediatric Inflammatory Bowel Disease

To establish an apparent diffusion coefficient (ADC) cut-off value to classify active and non-active lesions in inflammatory bowel disease

Spontaneous non-aortic retroperitoneal hemorrhage: etiology, imaging characterization and impact of MDCT on management. A multicentric study

Purpose: The purpose of this multicentric study is to assess the usefulness of multiphasic Computed tomography in the identification of spontaneous non-traumatic retroperitoneal hematoma (SRH) and its management, with references to the role of interventional radiology. Materials and methods: From January 2011 to June 2014, 27 patients with SRH were selected. Patients with aortic, traumatic, or iatrogenic source of bleeding were excluded. All the patients were studied with multiphasic MDCT after injection of intravenous contrast. Digital Subtraction angiography and percutaneous embolization treatment were performed. Results: CT identified SRH in all cases (100 %), showing the source of bleeding in 11 cases (40 %) and pointing out the source of bleeding in 15 cases (55 %). In one case (5 %), the bleeding origin was recognized only at surgery as adrenal source. CT has identified a contrast medium extravasation in the arterial phase in 17 patients (63 %), treated successfully by percutaneous embolization in 13 and by open-surgery in two cases. Two patients died before undergoing intervention and surgery, respectively. Ten patients (37 %) were non-operatively treated successfully with clinical, laboratory, and imaging follow-up. Conclusions: Multiphasic CT is the gold standard for the identification of a SRH. Recognition of CT signs of active bleeding is the crucial feature influencing the timing of therapeutic treatment. Urgent embolization should be performed in cases of arterial bleeding or contained vascular injuries supplying the retroperitoneal hematoma. Surgery is to be addressed in cases of actively bleeding hematomas associated with complication. Finally, an initial more conservative approach can be adopted in patients without signs of contrast extravasation or low-flow active bleeding. Technical skill, expertise, and recognition of CT signs of arterial active bleeding are critical features influencing patients management

Advances in Alport syndrome diagnosis using next-generation sequencing.

Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity

Risk factors for renal calcifications and determinants of hypercalciuria in patients with chronic, post-surgical hypoparathyroidism

Conventional therapy with oral calcium supplements and activated vitamin D is the most diffuse and available therapy for chronic hypoparathyroidism (HypoPT). This treatment does not replace the lack of PTH and is associated with renal complications. We report the results of a case control study with a prospective design which included 178 adult patients with differentiated thyroid cancer treated with total thyroidectomy with a follow-up longer that 3 years after surgery: 89 with PoHypoPT treated with conventional therapy and 89 without PoHypoPT, matched for age and sex. Both groups were balanced for gender, age, time since thyroidectomy, supplementation with cholecalciferol, dose of levothyroxine and dietary calcium intake. Half of the patients were stable on treatment with calcitriol alone, 45% with calcitriol and calcium carbonate, and 4 with calcium carbonate alone. All patients underwent biochemical tests and renal ultrasound. Twenty-fou- hour urinary calcium, creatinine, sodium, potassium, chloride, sulfate, uric acid, phosphate, oxalate, citrate, volume and Ph were measured. The biochemical control of patients with PoHypoPT was satisfactory, but only one-third of patients was at target according to ESE guidelines. Patients with PoHypoPT, compared with those without PoHypoPT, had significantly lower alb-Ca and PTH and increased serum phosphate, calcium-phosphate product, and 24-h urinary calcium, but there was no difference in estimated GFR. Renal calcifications were detected in 26 (29.2%) patients with PoHypoPT and in 11(12.4%) without. We found a positive association between renal calcification and age (P=0.03) and plasma PTH (P=0.01), but no association with hypercalciuria or other urinary parameters. The median 24-h urinary calcium was significantly higher in patients with PoHypoPT than in those without (248 vs 162 mg, P< 0.01) Urinary calcium in patients with PoHypoPT was positively associated with serum calcium (P <0.001), urinary magnesium (P <0.001), and urinary volume (P= 0.003), and negatively associated with serum albumin (P = 0.025), urinary oxalate (P <0.001) and creatinine (P= 0.008). Our study confirms that conventional therapy in patients with chronic PoHypoPT is suboptimal. 24-h urinary calcium and the rate of renal calcification are higher in patients with chronic PoHypoPT compared with controls. We found no significant difference in renal function (eGFR) between patients with chronic PoHypoPT compared with controls. We found no association between renal calcification and hypercalciuria and/or other urinary stone risk factors. Further prospective studies including a large number of patients would be necessary to better define the risk factor for renal calcifications in patients with PoHypoP

Circulating Coding and Long Non-Coding RNAs as Potential Biomarkers of Idiopathic Pulmonary Fibrosis

Background: Idiopathic Pulmonary Fibrosis (IPF) is a chronic degenerative disease with a median survival of 2–5 years after diagnosis. Therefore, IPF patient identification represents an important and challenging clinical issue. Current research is still searching for novel reliable non-invasive biomarkers. Therefore, we explored the potential use of long non-coding RNAs (lncRNAs) and mRNAs as biomarkers for IPF. Methods: We first performed a whole transcriptome analysis using microarray (n = 14: 7 Control, 7 IPF), followed by the validation of selected transcripts through qPCRs in an independent cohort of 95 subjects (n = 95: 45 Control, 50 IPF). Diagnostic performance and transcript correlation with functional/clinical data were also analyzed. Results: 1059 differentially expressed transcripts were identified. We confirmed the downregulation of FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) lncRNA, hsa_circ_0001924 circularRNA, utrophin (UTRN) and Y-box binding protein 3 (YBX3) mRNAs. The two analyzed non-coding RNAs correlated with Forced Vital Capacity (FVC)% and Diffusing Capacity of the Lung for carbon monoxide (DLCO)% functional data, while coding RNAs correlated with smock exposure. All analyzed transcripts showed excellent performance in IPF identification with Area Under the Curve values above 0.87; the most outstanding one was YBX3: AUROC 0.944, CI 95% = 0.895–0.992, sensitivity = 90%, specificity = 88.9%, p-value = 1.02 × 10−13. Conclusions: This study has identified specific transcript signatures in IPF suggesting that validated transcripts and microarray data could be useful for the potential future identification of RNA molecules as non-invasive biomarkers for IPF

The New Microtubule-Targeting Agent SIX2G Induces Immunogenic Cell Death in Multiple Myeloma

Microtubule-targeting agents (MTAs) are effective drugs for cancer treatment. A novel diaryl [1,2]oxazole class of compounds binding the colchicine site was synthesized as cis-restricted-combretastatin-A-4-analogue and then chemically modified to have improved solubility and a wider therapeutic index as compared to vinca alkaloids and taxanes. On these bases, a new class of tricyclic compounds, containing the [1,2]oxazole ring and an isoindole moiety, has been synthetized, among which SIX2G emerged as improved MTA. Several findings highlighted the ability of some chemotherapeutics to induce immunogenic cell death (ICD), which is defined by the cell surface translocation of Calreticulin (CALR) via dissociation of the PP1/GADD34 complex. In this regard, we computationally predicted the ability of SIX2G to induce CALR exposure by interacting with the PP1 RVxF domain. We then assessed both the potential cytotoxic and immunogenic activity of SIX2G on in vitro models of multiple myeloma (MM), which is an incurable hematological malignancy characterized by an immunosuppressive milieu. We found that the treatment with SIX2G inhibited cell viability by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, we observed the increase of hallmarks of ICD such as CALR exposure, ATP release and phospho-eIF2α protein level. Through co-culture experiments with immune cells, we demonstrated the increase of (i) CD86 maturation marker on dendritic cells, (ii) CD69 activation marker on cytotoxic T cells, and (iii) phagocytosis of tumor cells following treatment with SIX2G, confirming the onset of an immunogenic cascade. In conclusion, our findings provide a framework for further development of SIX2G as a new potential anti-MM agent

High frequency of COH1 intragenic deletions and duplications detected by MLPA in patients with Cohen syndrome

Cohen syndrome is a rare, clinically variable autosomal recessive disorder characterized by mental retardation, postnatal microcephaly, facial dysmorphisms, ocular abnormalities and intermittent neutropenia. Mutations in the COH1 gene have been found in patients from different ethnic origins. However, a high percentage of patients have only one or no mutated allele. To investigate whether COH1 copy number changes account for missed mutations, we used multiplex ligation-dependent probe amplification (MLPA) to test a group of 14 patients with Cohen syndrome. This analysis has allowed us to identify multi-exonic deletions in 11 alleles and duplications in 4 alleles. Considering our previous study, COH1 copy number variations represent 42% of total mutated alleles. To our knowledge, COH1 intragenic duplications have never been reported in Cohen syndrome. The three duplications encompassed exons 4–13, 20–30 and 57–60, respectively. Interestingly, four deletions showed the same exon coverage (exons 6–16) with respect to a deletion recently reported in a large Greek consanguineous family. Haplotype analysis suggested a possible founder effect in the Mediterranean basin. The use of MLPA was therefore crucial in identifying mutated alleles undetected by traditional techniques and in defining the extent of the deletions/duplications. Given the high percentage of identified copy number variations, we suggest that this technique could be used as the initial screening method for molecular diagnosis of Cohen syndrome