ANNUAL REPORT
Activity Report of Year I (cycle XXXIII)
One project is dedicated to understanding the role of micro-RNAs (miRNAs) in cancer cachexia or heart failure. This project is being carried out in collaboration with the Department of Clinical and Biological Sciences of the University of Turin (Prof. Paola Costelli). During this first year, after the approval of the ethics committee, 25 neoplastic patients (pancreas, stomach, and colon) and 15 healthy controls were enrolled. Cachexia was defined on the basis of: 1) hematochemical parameters, 2) bioimpedentiometry (BIA), 3) multilayer CT, and 4) specific questionnaires for the diagnosis of anorexia. From the plasma of the 40 subjects, exosomes and microvesicles were isolated. The total RNA extracted was subjected to a Next Generation Sequencing (NGS) method for miRnoma sequencing. The cytokines GDF15 and FGF21 which are involved in the pathogenesis of cachexia were also assayed in serum (ELISA). Preliminary results indicate that the levels of GDF15 and FGF21 are significantly higher in cancer patients than in healthy controls.
A second project aims to evaluate the expression of the Ki67 proliferation index and the epidermal growth factor receptor family, in particular HER2, in a series of differentiated thyroid carcinomas (DTC), with the aim of correlating the expression of these markers with the histotype and dimensions of the primitive neoplasm, multifocality, the presence of metastases, and the overall prognostic evaluation. The proliferative activity of Ki67 and the expression of epidermal growth factor receptors are determined by immunohistochemistry (IHC) using a specific monoclonal antibody. The presence of nuclei immunoreactive for Ki67 is evaluated with an automated technique using a light field microscope which quantifies the percentage by using special software. Expression of epidermal growth factor receptors, particularly HER2, is assigned a score from 0 to 3+. With a score of 2+, gene amplification is performed by the fluorescent in situ hybridization (FISH) technique. We retrospectively selected 113 patients (mean age: 50 years, 89 women and 24 men) who underwent total thyroidectomy from 2009 to 2017 and who had a histological diagnosis of DTC which was staged according to the TNM classification (AJCC VII 2009 edition). The 113 DTC patients were divided into two subgroups: 1) 109 patients with papillary carcinoma (PTC) (78 classic variant, 29 follicular variant, 1 oncocyte variant, 1 Warthin-like variant); and 2) four patients with follicular carcinoma (FTC). The Ki67 proliferative index is currently being analyzed in the first sample of 21 patients with classic papillary carcinoma.
Another project aims to clarify the molecular mechanism through which ANGPTL3 influences the expression of LDLR and LRP1. For this purpose, HepG2 cells were transfected using siRNA targeting human ANGPTL3 (siANGPTL3) or control siRNA (siCTRL). Through Western blot and RT-qPCR techniques, the effects on LDLR, LRP1, and PCSK9 protein and mRNA expression were evaluated. The siANGPTL3 cells showed higher levels of LDLR and LRP1 protein, but not mRNA, expression as compared to siCTRL. Since the PCSK9 protease regulates the degradation of both receptors, its expression was analyzed. Results showed lower PCSK9 protein expression, but no modulation of PCSK9 mRNA levels in siANGPTL3 cells as compared to control cells. These results demonstrate that ANGPTL3 modulates the expression of LRP1 and LDLR through the regulation of PCSK9 levels.
Another project involves the use of a network medicine approach in order to understand the reasons for vemurafenib response heterogeneity in lung (LUAD), thyroid (PTC), and colorectal cancer (CRC). SWItchMiner software (SWIM) was used to analyze gene expression profiles available from The Cancer Genome Atlas. SWIM identifies a small pool of regulatory genes (switch genes) which are likely to be associated with significant changes in the cellular phenotype. Then, using the Geneious R11 platform, we identified the switch genes that code for kinases and selected those with the greatest sequence homology to the known targets of vemurafenib. LUAD is the tumor with the highest number of switch genes (298) compared with the normal tissue, followed by PTC (227) and CRC (183). In these tumors, the switch genes that code for kinases are 14, 7 and 3, respectively. The three recurring motifs in the known vemurafenib targets are present in six switch genes coding for kinases in LUAD, and in five switch genes coding for kinases in PTC. In contrast, CRC has only one switch kinase with lower sequence homology. The presence of a different number of switch kinases, which vemurafenib targets, may explain the different drug response in the various tumors. However, this hypothesis must be validated with in vitro experiments.
The aims of the PhD project are to: 1) analyze the expression levels of AB receptors (FXR, LXR, TGR5 and PXR) in an intestinal (Caco2) and hepatic (HepG2) cell line, placed in co-culture and being overexpressed TNFalpha in murine models of non-alcoholic steatohepatitis (NASH) and colitis, in order to estimate the impact of intestinal inflammation on the liver and vice versa; 2) test the efficacy of innovative therapies, such as AB receptor agonists, glycyrrhetinic acid, omega-3, and probiotics in reducing intestinal and hepatic inflammation by acting on GLA. Preliminary data showed a decrease in the expression of inflamed FXR, LXR, PXR, and TGR5 receptors in Caco2 and HepG2. Preliminary trials of Caco2-HepG2 co-cultures have shown that the alteration of intestinal permeability during inflammation induces an increase in cytokines (IL-8 and IL1 beta) and a decrease in biliary receptors in HepG2.
Another project being undertaken by a foreign doctoral student from Iran evaluates the effects of ANGPTL3 on muscle cells. This study aims to improve understanding of the role of ANGPTL3 on the regulation of energy substrates. For this purpose, differentiated C2C12 cells were treated with ANGPTL3 (100nM) and the beta-agonist isoproterenol (100nM). Protein expression levels as well as mRNA levels of the genes involved in intracellular lipolytic pathways were analyzed at different times for a total of 90 minutes by Western blot and RT-qPCR analysis. Preliminary results indicated that treatment with ANGPTL3 and isoproterenol (ISO) reduce AMPK expression levels after 30 and 60 minutes, respectively. Cells treated with ANGPTL3, as well as those treated with ISO also showed an increase in the gene and protein expression of ERK1/2, as well as their phosphorylated form.
In another project, we wanted to evaluate the effect of high-dose allopurinol (300 mg / day) on the progression of hs-PCR values and on platelet aggregation in patients with acute myocardial infarction (AMI) and incomplete myocardial revascularization as compared to controls. In this prospective, monocentric cohort study, 116 patients with AMI undergoing pharmacological treatment and 116 control patients were enrolled. For all the patients enrolled, we collected anamnestic data and the angiographic localization of the "culprit lesion". The hs-PCR levels, as well as the platelet reactivity, expressed in terms of PRU, were determined at three different times (T0: basal; T1: after 30 days from the start of allopurinol 300 mg; T2: after another 30 days of allopurinol; T3: after 5 days of allopurinol wash-out). Preliminary data show a non-significant reduction in hs-PCR levels (p = NS) in both control patients and patients treated with allopurinol. However, allopurinol determined a significant reduction in platelet reactivity in treated patients compared to control cases: (T1 vs T2: p = 0.0001; T2 vs T3: p = 0.0001).
The project aims to analyze the factors that can influence the progression of bladder carcinoma from the initial state (dimension below 200-300 μm), still well localized, towards the malignant tumor phenotype with all the invasive and metastatic biological characteristics (uncontrolled growth, ability to invade adjacent tissues). Eighteen patients with bladder cancer were selected: four women and 14 men, aged between 40 and 85 years. In each case, at least three tissue samples were collected during surgery. The cryopreserved samples were analyzed with the LCMD (laser capture microdissection) technique in order to perform a cell-specific analysis. RNA was extracted from the cells using the EZ1 biorobot. The RNA obtained will be retrotranscribed and the cDNA obtained will be subjected to real-time PCR on customized low-density arrays, in 50 ml of a PCR Master Mix. The same cDNA will be analyzed using microfluidic cards customized with genes of interest (including HIF-1, NFkB, iNOS2, RAGE, P2X7R). At the moment only the histological analyses have been completed.
The goal of another doctoral project is to identify new biomarkers circulating in medullary thyroid carcinoma (MTC). Patients with sporadic/hereditary MTC will be prospectively enrolled in the study: cohort-1 (newly diagnosed patients awaiting surgery on the primary tumor); cohort-2 (patients with persistent disease, eligible for local/systemic treatment). In order to search for circulating biomarkers (miRNA, circulating free DNA - cfDNA) tumor tissue (primitive and/or secondary) and plasma (at baseline and after treatment) will be analyzed for each patient. The analysis of miRNA expression will be performed both at the tissue level and through PCR. Mutational analysis of tumor tissue and plasma will be performed using the NGS technique. The identified mutations will be validated and monitored over time using digital-PCR. Patients will be stratified according to treatment response.
In 2018, first year activities of a prospective longitudinal project to assess the relationship between microRNA and renal fibrosis in autosomal dominant polycystic kidney disease (ADPKD) were resumed. During this time, the study enrolled 25 ADPKD patients. A total of 15 patients with different degrees of renal function underwent laboratory tests, MR at 3T, for the quantification of total kidney (TKV), perfusional (TPV), and fibrotic volume (TFV). Preliminary data indicate that TKV (<0.02), TPV / TKV% (p <0.02) and TFV (p <0.04) were negatively and significantly correlated with eGFR (EPI). TFV was positively and significantly correlated with plasma aldosterone (p <0.05), insulinemia (p <0.05), and ABI (p <0.05).
Activity Report of Year II (cycle XXXII)
This project is dedicated to investigating the clinical and therapeutic aspects of rheumatological manifestations in a cohort of pediatric patients with chronic inflammatory bowel disease (IBD). In the second year of this study 83 patients with IBD and articular manifestations were enrolled (56 Crohn’s disease, 27 ulcerative colitis). The prospective evaluation of these patients showed that arthritis had an earlier onset than arthralgia (p = 0.031), with no difference in prevalence of arthritis or arthralgia in Crohn’s disease vs. ulcerative colitis (p = 0.930). The frequency of small, coxofemoral, and sacroiliac joint involvement was greater in patients with arthritis (p = 0.006, p = 0.001, p = 0.001, respectively). In Crohn’s disease, in both groups with arthritis and arthralgia, ileocolon (L3) involvement was greater than that of the ileum (L1) and colon (L2). The remission of arthritis with top down vs. step up therapy has already shown significant differences at six (p = 0.043) and 12 months (p = 0.036).
This project, dedicated to the definition of the role of NBS1, N-MYC, and the Sonic Hedgehog (SHH) pathway in cerebellar development and carcinogenesis continued in its second year with the development of two animal models (NbnGCP-Δ and NBNfl / flGli1 / CRE-tamoxifen- driven). The study of the murine model NbnGCP-Δ obtained from the conditional deletion of Nbs1 in cerebellar granule cell progenitors (GCPs) shows how the absence of Nbs1 determines cerebellar development defects and the downregulation of the SHH pathway in ex vivo GCPs. The downregulation of the pathway was also confirmed in-vitro in neurospheres/GCP-NBNfl/flGli1/CRE-tamoxifen-driven. Among the possible causes is the role of Nbs1 in the regulation of primary ciliogenesis, an essential process of the SHH pathway. Our data show that the absence of Nbs1 results in a morphological alteration of the primary cilium ex-vivo and in-vitro, and in ciliary disassembly abnormalities.
In its second year, this project, dedicated to the assessment of respiratory microbiota and immune response in infants hospitalized for bronchiolitis, has enrolled 40 children aged <6 months who were subjected to nasopharyngeal aspirate in order to search for 14 respiratory viruses. Metagenomic DNA was then extracted with subsequent sequencing of the bacterial communities. The patients were divided into two groups: virus positive (21) and virus negative (10). In nine cases it was not possible to extract DNA from the collected biological material. Less richness and biodiversity were observed in terms of bacterial species in the presence of respiratory viruses. Streptococcus pseudopneumoniae was discriminating and was present more often (p = 0.013) in children with respiratory virus. Other discriminatory species for respiratory viruses are Staphylococcus epidermidis (p = 0.048), Rubrobacter xylanophilus (p = 0.025), and Corynebacterium tuberculostearicum (p = 0.025).
This project is dedicated to the identification and characterization of Hedgehog pathway antagonistic drugs for the treatment of brain tumors. In the second year results demonstrated the antitumor activity of small molecules, identified at the beginning of this project, which were able to block the Hh pathway acting both upstream on the Smo receptor, and downstream on the Gli1 transcription factor. Having also identified a new positive regulator of signaling, the transcription factor Sall4, which interacts with HDAC1 (a known activator of Hh), the research conducted has defined some molecular details regarding the regulation of the transcriptional activity of Gli1.
This project is aimed at determining the effects of dipotassium glycyrrhizinate (DPG) on the regulation of mucosal healing (MH) genes in the intestinal epithelium following an acute phase of experimentally induced colitis in murine models. In the second year, this study reported that DPG accelerates MH by inhibiting pro-inflammatory genes (CXCL1, CXCL3, CXCL5, PTGS2, IL-1β, IL-6, CCL12, CCL7) and activating central genes of the MH pathway (PLAUR, VTN, COL3A1, MMP9, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1). In particular, during inflammation PLAUR and VTN are fundamental in vitro for efficient repair and functionality of the intestinal mucosa, as mediated by DPG.
This project compares the intestinal microbiota of children with an allergy to cow's milk protein (APLV) with that of healthy subjects of the same age. For this comparative study three groups of children were enrolled. Group 1 included children with a sensitization to cow's milk (positivity of skin prick test and specific IgE for PLV, cut-off 0.35 kUI / L) and positivity to an oral challenge test with double-blind placebo-controlled cow milk (DBPCFC). Group 2 included children with sensitization to PLV, but DBPCFC negative. Group 3 included healthy children with a specific IgE for negative PLV. In its second year, this study reported the selection of 33 patients (16 females, median age 12.8 months, range 11-15 months). Of these patients, 12 were included in Group 1, six in Group 2, and 15 in Group 3. The microbiota analysis showed that Bifidobacterium longum BB536 and Bifidobacterium breve M-16V were similarly present among the three groups at a mean concentration of 104-105 cells / ul, whereas bifidobacterium infantis M-63 was completely absent in the three groups.
The project, dedicated to the evaluation of the role of oxidative stress in thyroid disease, in its second year continued the enrollment of patients with thyroid tumors cytologically diagnosed as Thy3b, Thy4, and Thy5 who are candidates for total thyroidectomy and lymphectomy (n = 20). The expression of GPx1 and TrxR1 was determined by Western blot on samples of thyrocytes taken from both the tumor nodule and healthy tissue, while the evaluation of oxidative stress was performed using the EPR-spin trapping technique. Preliminary results showed a significant decrease in the expression of GPx1 and TrxR1 (-45.7% and -43.2%, respectively, p <0.01) in thyroid tumor tissue compared to healthy tissue, whereas EPR demonstrated an increase in free radicals in the tumor as compared to healthy thyrocytes (+ 38.7% a.u. p <0.01).
An experimental oncogenesis project was dedicated to the evaluation of polyamines (spermine, spermidine, putrescine and cadaverine) in cell proliferation and cancer. The main regulator of their metabolism is ornithine decarboxylase (ODC). High levels of polyamine and ODC are found in many tumors and the pharmacological inhibition of this axis (DFMO, ODC inhibitor) has an antitumor effect in vitro and in vivo. Despite this, the cell activates rescue mechanisms of the polyamines from the external environment and this makes it necessary to act on downstream effectors of the pathway. The translational factor Eif5a, over-expressed in many tumors, is activated by the posttranslational modification of DHPS and DOHH-mediated hypusination using spermidine as a substrate. During the second year of this project, the effect of DHPS inhibitor GC7 on the proliferation of colorectal carcinoma (CRC) cell lines (HCT116, HT29, and LoVo) was evaluated. The results indicate that inhibition of the hypusine axis reduces the proliferation of CRC cells and c-Myc protein levels in vitro and in vivo. GC7 showed a marked synergistic effect in vitro in combination with DFMO.
Activity Report of Year III (cycle XXXI)
This project aimed to evaluate in vitro the effects of different compounds that target different mutations involved in the development and progression of prostate cancer (PCa) and compare these results with the clinical experience of therapy response in PCa patients with different progression and expression of AR-V7 in the nucleus. For this study several cell lines were used: 1) LnCaP (which maintains the sensitivity to androgens, expresses PSA and PSMA, maintains WT p53 and presents mutated PTEN that leads to the constitutive activation of the AKT pathway); and 2) VCaP cells derived from vertebral metastases isolated from a lumbar vertebra of a patient refractory to hormone therapy. These cells express PSA and PAP (prostatic acid phosphatase). VCaP cells express wt AR but present an amplification of AR which results in higher levels of AR-FL. They also present AR splice variant-7 (AR-V7). They express PTEN and the TMPRSS2-ERGPTEN fusion gene. In these cells the PI3K / AKT/ mTOR, Wortmannin, and RAD001 inhibitors were evaluated in combination with drugs that interfere with the AR pathway. Docetaxel was found to have a greater ability to inhibit cell growth of both cell lines. After treatment with 5nM, only 14% and 30% of LnCaP and VCap cells continued to proliferate, respectively. Treatment with Abiraterone reduced the growth of LnCap cells by only 40% and that of VCaP by 20%, while Enzalutamide reduced the growth of LnCAP by 55% and VCap by 30%. Treatment of these cells with PI3K / AKT / mTor inhibitors effectively reduced the survival of the examined cells.
This research project foresees the creation of multiparametric scores in order to determine the estimated malignancy risk of thyroid nodules subjected to fine needle cytology. A total of 917 nodules were sampled, of which 82 were excluded for a size <1 cm and 282 for the absence of a conclusive diagnosis. The sonographic characteristics of the nodules were recorded and used to classify each nodule according to the American Association of Clinical Endocrinologists (AACE / ACE / AME), American College of Radiology (ACR), American Thyroid Association (ATA), EU-TIRADS, and K-TIRADS guidelines. The reference standard was the definitive histology if available or a benign cytology on subsequent follow-up. Non-diagnostic or undetermined cytology was excluded. The application of a classification system would avoid 92 (16.6%) to 287 (51.9%) needle aspirates (K-TIRADS and ACR TIRADS, respectively [p <0.001], with a false-negative rate of 3.3% and 2.8 %). The malignancy rate in the various categories was consistent with the estimated risk. In summary, sonographic stratification allowed a better selection of candidate nodules for cytology and molecular analysis, through the pre-test estimation of malignancy and optimized predictive values.
The aim of this study was to evaluate cardiac function in subjects affected by del22q11 syndrome without CC, as compared with a control group. The study involved 20 adult subjects with del22q11 without CC and a control group of 14 healthy subjects. All enrolled subjects underwent transthoracic color-doppler echocardiography. The ultrasound study showed that patients with del22q11.2 had normal values. However, in the del22q11 group, statistically significant changes were recorded for the M-mode values of the interventricular septum and left ventricular mass indices and, using Tissue Doppler Imaging, there were variations of some transmitral flow values which may suggest a diastolic dysfunction in the subclinical phase. Patients with del22q11 have a substantially normal ventricular function. However, significant differences between the two groups were found for some values, which should be further investigated to understand the cause and set up follow-up programs.
The aim of this study was to identify clinical and molecular prognostic factors in differentiated thyroid carcinoma (DTC) using a national prospective and multicentric database. Recurrence risk and response to treatment were classified according to the American Thyroid Association (ATA) guidelines. In 721 patients, a follow-up of ≥3 years was achieved. The recurrence risk was low in 417 (57.8%) patients, intermediate-low in 143 (19.8%), intermediate-high in 128 (17.8%), and high in 33 (4.6%). Treatment response was excellent in 452 (61.8%), biochemistry incomplete in 36 (4.7%), indeterminate in 198 (27.3%), and structural incomplete in 44 (6.2%). Patients with an intermediate-high and high risk had a higher rate of structural disease at three years than patients with low risk (15% and 21% vs. 3%, respectively). The time profile of miR-146a-5p and miR-221-3p was analyzed in 44 patients. There was a decreasing trend in disease-free patients and an increasing trend in patients with disease persistence. The ATA risk classification is effective in predicting the structural persistence of the disease. One third of patients present an indeterminate disease status with current markers. In these cases, the miRNA profile is a promising alternative.
The main purpose of this study was to identify and quantify the energy substrates used by the heart in moderate-to-severe SC. For this aim, after at least 6 hours of fasting, blood concentrations of β-hydroxybutyrate, lactate, triacylglycerols, glucose and fatty acids both in the arterial and in the coronary venous circle were measured simultaneously in patients who were candidates for the implantation of a cardiac device. A total of 16 patients with SC and 15 controls were enrolled. The energy substrate concentrations in both the arterial and venous sides were similar in the two groups, whereas their arterio-venous difference showed a significant reduction in fatty acid extraction in patients with SC (SC 0.070.23 mmol / L vs non-SC 0.250.16 mmol / L, p = 0.03). Even when only nondiabetic patients were analyzed, the results were substantially unchanged (SC 0.030.30 mmol/L vs non-SC 0.260.12 mmol / L, p = 0.09). In this study the use of ketone bodies by the heart muscle was superimposable in patients with heart failure and controls. On the contrary, a reduction in fatty acid extraction was demonstrated in subjects with SC, compatible with a down regulation of beta-oxidation which has already been demonstrated in more advanced stages of the disease.
One project was dedicated to the prospective evaluation of the impact of sarcopenia on the outcome of patients with lung cancer, in relation to the Glasgow Prognostic Score (GPS). An additional objective was to correlate the presence of sarcopenia with the values of GDF 15 and miRNA. Of the 35 patients enrolled, 18 are sarcopenic and 17 are non-sarcopenic; 12 patients are classified in GPS 0, 15 patients in GPS 1, and 8 patients in GPS 2. GPS 0 patients are predominantly non-sarcopenic whereas GPS 2 patients are sarcopenic (p = 0.023). A statistically significant correlation between GPS and nutritional status was found (p = 0.0013). Sarcopenic and anorexic patients showed significantly higher PCR values. Chemotherapy was well tolerated: 12 patients presented dose-limiting toxicity (DLT). The data obtained show an association between sarcopenia, anorexia, and malnutrition. The presence of sarcopenia and malnutrition is associated with a worse prognostic index. The degree of sarcopenia and anorexia is related to increased inflammation rates. Sarcopenic patients show more toxicity than non-sarcopenic patients.
One project aimed to determine biventricular function patterns and their relationship with indicators of pre- and postnatal clinical severity in infants with congenital diaphragmatic hernia (CDH). The microRNA cluster miRNA 17-92, typical of pulmonary hypoplasia, was evaluated in patients with CDH. The study was conducted as an observational case-control study in patients with CDH. Early cardiac function was evaluated by speckle tracking-derived global strain and tissue Doppler imaging. Research regarding miRNA 17-92 was performed with bronchoalveolar lavage. Subsequently this cluster was correlated with the Lung Head Ratio (LHR), the oxygenation index (OI), the duration of intubation (DINT), and the length of hospital stay (LOS). All functional measurements were significantly reduced in patients with CDH (n = 25) compared to the control group (n = 20) within 48 hours of life. MiR17, miE18a, miR19b, and miR20a were found to be significantly elevated in patients with CDH (p <0.05). Only left ventricular dysfunction showed a positive correlation with LHR (r2 = 0.32, p=0.03), with OI (r2 =0.35, P <0.001), with DINT (r2 =0.24, p=0.04), and with LOS ( r2 = 0.4, p=0.006). The transitional period in CDH is characterized by biventricular cardiac dysfunction. Left ventricular function also correlates with the clinical markers of pre and postnatal severity and therefore appears to have a determining role in the severity of this pathology, and also represents a possible therapeutic target. These results support the importance of regular assessment of cardiac function. MiR 17-92 may represent an important marker of pulmonary hypertension in patients with CDH.
One project evaluated early neuroradiological markers of neurodegeneration in children with multiple sclerosis (MS) and clinically isolated syndrome (CIS). A total of 62 patients (20 MS, 42 CIS) were recruited. The lesion load at onset was higher in MS vs. CIS patients (14.1 ± 12 vs 9.5 ± 7, p <0.05). As compared to controls, MS and CIS patients showed a reduction in total cerebral volume (1.56 x 106 vs 1.64 x 106, p <0.05). Spectroscopic analysis showed an increase in glutamate levels (8.1 ± 1.6) in apparently normal white matter in CIS/MS patients compared to controls, as well as an increase in glutamate (8.0 ± 1) in gadolinium-enhanced lesions and a reduction in N-acetyl-aspartate (10.8 ± 2) in chronic lesions. There is a relationship between cognitive impairment and the volume of cortical lesions (r = 0.59; P .001), hyperintense lesions in T2 (r = 0.41; P .001), and volume of gray matter (r = -0.47; P .001). Logistic regression showed that reduced cognitive performance correlates with the number of cortical lesions (r = 0.59; P .001), the volume of hyperintense lesions in T2 (r = 0.41; P .001) and the volume of gray matter (r = -0.47; P .001). After a 12-month follow-up, it was observed that patients who showed a significant reduction in cortical volume from the onset showed greater disease aggressiveness (number of recurrences and decreased cognitive performance).
Based on the premise that atrial fibrillation (AF) is usually associated with multiple comorbidies which can be evaluated by the Charlson Comorbidity Index (CCI), a validated tool for the assessment of comorbidity load, this research projects aims to study the relationship between AF and CCI through the use of a large administrative database of the Lombardy region (> 10 million inhabitants). All admitted patients diagnosed with AF in 2002 were considered for analysis and followed up for 12 years. These patients were compared with a random sample of subjects without AF. The diagnosis of AF and the construction of the CCI were evaluated using the ICD-9 codes obtained from hospitalizations. In 2012, 24,040 patients were admitted with a diagnosis of AF and had a significantly higher CCI compared to 24,0400 patients without AF (1.8 ± 2.1 vs. 0.2 ± 0.9, p <0.001). Over the 12 years of observation, an adjusted linear model showed that AF was independently associated with a risk of significant increase in the CCI (beta coefficient: 1.69, 95% confidence interval [CI]: 1.67-1.70). Adjusted logistic regression analysis showed that the CCI was inversely associated with the prescription of anticoagulants at baseline (OR: 0.91, 95% CI: 0.89-0.92 for each point) and at the end of observation (OR: 0.98, 95% CI: 0.98 -0.99). During the 12 years of observation, AF patients with a high CCI (≥4) had a higher cumulative incidence of stroke, major bleeding, and death (all p <0.001), compared to AF patients with a low CCI (0-3). In this cohort study, AF was independently associated with an increased risk of a progressive increased comorbidity load, assessed as CCI. An increased CCI was inversely associated with oral anticoagulant prescription throughout the observation period. Lastly, CCI was independently associated with an increased risk of stroke, major bleeding, and death.
The aim of the project was to validate the use of Next Generation Sequencing (NGS) for the simultaneous analysis of multiple genes in predictive and prognostic oncology diagnostics. The presence of germinal mutations in patients with hereditary breast/ovarian cancer (HBOC) and somatic mutations in cases of metastatic melanoma (MM) were investigated. For this purpose, 152 cases of HBOC were analyzed to identify germline mutations in BRCA1/2 high penetrance genes. A total of 26 cases of BRCA1/2 negative results were analyzed with a custom panel for the analysis of 29 genes associated with the risk of developing HBOC. The profile of somatic mutations in 22 key genes of tumorigenesis was analyzed in 46 cases of MM through the use of a commercial panel. Approximately 24% (37/152) of HBOC cases were carriers of mutations in BRCA1/2. Approximately 11% (3/26) of the negative BRCA 1/2 cases had pathogenetic mutations in the ATM, MUTYH, and PALB2 genes whereas 61% (16/26) had 20 variants of uncertain significance in the genes MSH6, APC, TP53, RAD51B, EPCAM, BRIP1, CHEK2, RAD50, STK11, and BARD1. The analysis of MM cases showed that 48% of cases had BRAF mutations and 33% of cases had NRAS mutations. The results of the study highlight the usefulness of NGS technology in oncological diagnostics in order to obtain the best prognostic stratification.
During 2018, four research projects of doctoral students belonging to previous cycles were resumed and completed. The first, dedicated to identifying therapeutic targets in cancer stem cells of colorectal carcinoma (CR-CSC), reported that a combination therapy of anti-HER2 and PI3K and BRAF inhibitors is able to determine a partial therapeutic response in BRAF-mutated tumors and a slowed tumor progression in tumors with KRAS mutations. Furthermore, a significant and persistent decrease in tumor mass was shown in a model of combined therapy in which anti-BRAF drugs were replaced with MEK inhibitors that cause down regulation of MICs in CR-CSC.
The second project, aimed at evaluating the therapeutic efficacy of the top-down approach (early introduction of biological drugs) and comparing it with the step-up approach (conventional therapy), showed in a group of 49 children with Crohn’s disease that an early introduction of biologic drugs was more effective in achieving complete mucosal healing, longer steroid-free periods, and lower recurrence rates.
Another project reported data from the CD-GEMM study (Celiac Disease Genetic Enviromental Microbiome and Metabolome), a prospective study conducted simultaneously in the USA and Italy that attempted to evaluate the contribution the metagenome to genetic risk of celiac disease through a modern approach to systems biology. From the heat maps related to the presence and abundance of genus and species for each initial sample collection period (7 days of life, 3 months, 6 months) a segregation was observed into two clusters (infants born through Cesarean/formula-fed/exposed to antibiotics at birth vs. infants born after vaginal delivery/breastfed/ without exposure to antibiotics at birth). An additional cluster segregation was also recorded with regard to metabolomic patterns depending on the genetics of the studied children.
The last of these projects, aimed at identifying preoperative prognostic factors able to stratify the risk of central compartment metastasis in papillary thyroid carcinoma. This study looked at 47 consecutive patients affected by papillary carcinoma of the unifocal thyroid and without clinical evidence of lymph node metastasis (cN0) and found that there were no preoperative parameters able to identify a population "at risk" of lymph node involvement.