Sviluppo di una micro CT con sorgente Quasi-Monocromatica Multi-Energy per lo studio in vivo della crescita e della metastasi tumorale

Un innovativo micro scanner CT per piccolo animali – basato su di una sorgente che genera una coppia di fasci X Quasi-Monocromatici paralleli con diverse energie selezionabili – è in corso di installazione e caratterizzazione al Dipartimento di Fisica dell’Università di Bologna. Lo scopo della ricerca è quello di effettuare l’imaging radiologico in vivo del tessuto tumorale e/o dei pattern di neo-angiogenesi in una fase diagnostica precoce realizzando la separazione del tessuto patologico da quello sano per mezzo della tecnica multi-energy che consiste nell’utilizzo di due o più fasci di raggi X quasi-monocromatici in sostituzione dell’unico fascio policromatico utilizzato nella radiologia convenzionale. Lo strumento consentirà inoltre lo studio, sui topi, della crescita tumorale e della formazione delle metastasi per differenti tipologie di tumore. Per la diagnosi precoce del tumore è essenziale essere in grado di rivelare i cambiamenti tissutali precancerosi, come la neo-angiogenesi. Si tratta di un meccanismo che si verifica in una fase iniziale dello sviluppo della patologia ed è dovuto alla produzione di molecole che stimolano la creazione di nuovi vasi sanguigni per alimentare la crescita delle cellule cancerose. Come dimostrato in precedenti studi di fattibilità [1], un sistema di imaging basato su due fasci di raggi X quasi-monocromatici di differenti energie fornisce maggiore sensibilità nella rivelazione di basse concentrazioni di mezzo di contrasto iodato rispetto ai tradizionali apparati RX con fascio policromatico. La K-edge dual energy radiology è una tecnologia potenzialmente in grado di rivelare il processo di neo-angiogenesi tumorale in uno stadio precoce quando la strumentazione convenzionale non dispone di sufficiente sensibilità. Inoltre, la possibilità di selezionare le energie dei fasci quasi-monocromatici consente l’applicazione della Multi-Energy Quasi-MonochromaticRadiology: selezionando opportunamente le energie è possibile esaltare le differenze fra i coefficienti di attenuazione lineare del tessuto patologico rispetto a quello sano aumentando il contrasto della patologia. Infatti, la tecnica multi-energy consente di ricostruire il numero atomico efficace e persino la composizione chimica del tessuto irradiato. Tuttavia, per ottenere questo risultato, si dovrebbero conoscere le bande di energia in cui l’assorbimento dei raggi X da parte del tessuto tumorale eventualmente differisce significativamente da quello dei tessuti sani. Per questo motivo è stata iniziata una sistematica caratterizzazione radiologica di molti tipi di tessuti sani e neoplastici, murini e umani allo scopo di costituire un catalogo delle finestre di energia in cui sarà possibile applicare la metodica multi-energy

Long-term physical training and left ventricular remodelling after anterior myocardial infraction: Results of the excercise in anterior myocardial infraction (EAMI) trial

AbstractObjectives. The aim of this multicenter randomized study was to investigate whether long-term physical training would influence left ventricular remodeling after anterior myocardial infarction.Background. Exercise is currently recommended for patients after myocardial infarction; however, the effects of long-term physical training on ventricular size and remodeling still have to be defined.Methods. Patients with no contraindications to exercise were studied 4 to 8 weeks after anterior Q wave myocardial infarction and 6 months later by echocardiography at rest and bicycle ergometric testing. After the initial study, patients were randomly allocated to a 6-month exercise training program (n = 49) or a control group (n = 46). A computerized system was used to derive echocardiographic variables of ventricular size, function and topography.Results. After 6 mongths, a significant (p < 0.01) increase in work capacity (from 4,596 ± 1,246 to 5,508 ± 1,335 kp-m) was observed only in the training group, whereas global ventricular size, regional dilation and shape distortion did not change in either the control or the training group. However, compared with patients with an ejection fraction >40%, patients with an ejection fraction ≤ 40% had more significant (p < 0.001) ventricular enlargement at entry and demonstrated further (p < 0.01) global and regional dilation after 6 months, in both the control and the training, group (end-diastolic volume from 77 ± 14 to 85 ± 17 ml/m2in the control group and from 74 ± 11 to 77 ± 15 ml/m2in the training group; regional dilation from 46 ± 18% to 57 ± 21% in the control group and from 42 ± 18% to 44 ± 26% in the training group). Ventricular size and topography did not change in patients with an ejection fraction >40%.Conclusions. Patients with poor left ventricular function 1 to 2 months after anterior myocardial infarction are prone to further global and regional dilation. Exercise training does not appear to influence this spontaneous deterioration. Thus, postinfarction patients without clinical complications, even those with a large anterior infarction, may benefit from long-term physical training without any additional negative effect on ventricular size and topography

Bioprofiling TS/A Murine Mammary Cancer for a Functional Precision Experimental Model

The TS/A cell line was established in 1983 from a spontaneous mammary tumor arisen in an inbred BALB/c female mouse. Its features (heterogeneity, low immunogenicity and metastatic ability) rendered the TS/A cell line suitable as a preclinical model for studies on tumor-host interactions and for gene therapy approaches. The integrated biological profile of TS/A resulting from the review of the literature could be a path towards the description of a precision experimental model of mammary cancer

Technetium-99m sestamibi tomographic evaluation of residual ischemia after anterior myocardial infarction

Objectives.This study investigated the value of sestamibi scintigraphy in assessing residual ischemia after anterior myocardial infarction.Background.Serial imaging with sestamibi, the uptake and retention of which correlate with regional myocardial blood flow and viability, has been used to estimate salvaged myocardium and risk area after acute infarction. We recently documented that recovery of perfusion and contraction in the infarcted area may continue well after the subacute phase, suggesting myocardial hibernation. Some underestimation of viability in the setting of hibernating myocardium by sestamibi imaging has been reported.Methods.We studied 58 patients in stable condition after Q wave anterior infarction. Regional perfusion and function were quantitatively assessed by sestamibi tomography and two-dimensional echocardiography at 4 to 6 weeks and at 7 months after infarction. In sestamibi polar maps, abnormal areas with tracer uptake >2.5 SD below our reference values were computed at rest and after symptom-limited exercise. On two-dimensional echocardiography the ejection fraction and extent of rest wall motion abnormalities were assessed by a computerized system. All patients had coronary angiography between the two studies.Results.At 7 months the extent of rest sestamibi defect was significantly reduced in 40 patients (69%, group 1) and unchanged in 18 (31%, group 2). Rest wall motion abnormalities and ventricular ejection fraction significantly improved in group 1 but not in group 2. Underlying coronary disease, patency of the infarct-related vessel and rest sestamibi defect extent at 5 weeks were comparable between the two groups. At 7 months, an increase in the reversible (stress-rest defect) tracer defect was observed in group 1 (p < 0.05) despite a smaller stress-induced hypoperfusion (p < 0.05). Reversible sestamibi defects and stress hypoperfusion were unchanged in group 2. In 38 (95%) of 40 group 1 patients, the area showing reversible sestamibi defects at 7 months matched the area showing fixed hypoperfusion at 5 weeks.Conclusions.The reduction in the rest tracer uptake defect that can occur late after infarction may affect the assessment of ischemic burden by sestamibi imaging early after anterior myocardial infarction

Evolution of HER2-positive mammary carcinoma: HER2 loss reveals claudin-low traits in cancer progression

HER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2labile cell line gave rise to HER2-negative tumors from which MamBo38HER2loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2labile cells induced the loss of HER2 expression. MamBo38HER2loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss

Vaccines against human HER2 prevent mammary carcinoma in mice transgenic for human HER2

INTRODUCTION: The availability of mice transgenic for the human HER2 gene (huHER2) and prone to the development of HER2-driven mammary carcinogenesis (referred to as FVB-huHER2 mice) prompted us to study active immunopreventive strategies targeting the human HER2 molecule in a tolerant host. METHODS: FVB-huHER2 were vaccinated with either IL-12-adjuvanted human HER2-positive cancer cells or DNA vaccine carrying chimeric human-rat HER2 sequences. Onset and number of mammary tumors were recorded to evaluate vaccine potency. Mice sera were collected and passively transferred to xenograft-bearing mice to assess their antitumor efficacy. RESULTS: Both cell and DNA vaccines significantly delayed tumor onset, leading to about 65% tumor-free mice at 70 weeks, whereas mock-vaccinated FVB-huHER2 controls developed mammary tumors at a median age of 45 weeks. In the DNA vaccinated group, 65% of mice were still tumor-free at about 90 weeks of age. The number of mammary tumors per mouse was also significantly reduced in vaccinated mice. Vaccines broke the immunological tolerance to the huHER2 transgene, inducing both humoral and cytokine responses. The DNA vaccine mainly induced a high and sustained level of anti-huHER2 antibodies, the cell vaccine also elicited interferon (IFN)-gamma production. Sera of DNA-vaccinated mice transferred to xenograft-carrying mice significantly inhibited the growth of human HER2-positive cancer cells. CONCLUSIONS: Anti-huHER2 antibodies elicited in the tolerant host exert antitumoral activity

Bone sarcoma patient-derived xenografts are faithful and stable preclinical models for molecular and therapeutic investigations

Standard therapy of osteosarcoma (OS) and Ewing sarcoma (EW) rests on cytotoxic regimes, which are largely unsuccessful in advanced patients. Preclinical models are needed to break this impasse. A panel of patient-derived xenografts (PDX) was established by implantation of fresh, surgically resected osteosarcoma (OS) and Ewing sarcoma (EW) in NSG mice. Engraftment was obtained in 22 of 61 OS (36%) and 7 of 29 EW (24%). The success rate in establishing primary cell cultures from OS was lower than the percentage of PDX engraftment in mice, whereas the reverse was observed for EW; the implementation of both in vivo and in vitro seeding increased the proportion of patients yielding at least one workable model. The establishment of in vitro cultures from PDX was highly efficient in both tumor types, reaching 100% for EW. Morphological and immunohistochemical (SATB2, P-glycoprotein 1, CD99, caveolin 1) studies and gene expression profiling showed a remarkable similarity between patient's tumor and PDX, which was maintained over several passages in mice, whereas cell cultures displayed a lower correlation with human samples. Genes differentially expressed between OS original tumor and PDX mostly belonged to leuykocyte-specific pathways, as human infiltrate is gradually replaced by murine leukocytes during growth in mice. In EW, which contained scant infiltrates, no gene was differentially expressed between the original tumor and the PDX. A novel therapeutic combination of anti-CD99 diabody C7 and irinotecan was tested against two EW PDX; both drugs inhibited PDX growth, the addition of anti-CD99 was beneficial when chemotherapy alone was less effective. The panel of OS and EW PDX faithfully mirrored morphologic and genetic features of bone sarcomas, representing reliable models to test therapeutic approaches

Resources and tools for rare disease variant interpretation

: Collectively, rare genetic disorders affect a substantial portion of the world's population. In most cases, those affected face difficulties in receiving a clinical diagnosis and genetic characterization. The understanding of the molecular mechanisms of these diseases and the development of therapeutic treatments for patients are also challenging. However, the application of recent advancements in genome sequencing/analysis technologies and computer-aided tools for predicting phenotype-genotype associations can bring significant benefits to this field. In this review, we highlight the most relevant online resources and computational tools for genome interpretation that can enhance the diagnosis, clinical management, and development of treatments for rare disorders. Our focus is on resources for interpreting single nucleotide variants. Additionally, we present use cases for interpreting genetic variants in clinical settings and review the limitations of these results and prediction tools. Finally, we have compiled a curated set of core resources and tools for analyzing rare disease genomes. Such resources and tools can be utilized to develop standardized protocols that will enhance the accuracy and effectiveness of rare disease diagnosis

HER2 cancer vaccine optimization by combining Drosophila S2 insect cell manufacturing with a novel VLP-display technology

Breast cancer is a widespread oncology indication affecting more than 1.3 million people worldwide annually, 20%-30% of which are HER2 positive. HER2 is a tyrosine kinase receptor that is frequently overexpressed in several solid-tumor cancers (incl. breast, prostate, gastric, esophageal and osteosarcoma) where it denotes an aggressive phenotype, high metastatic rate, and poor prognosis. In a human context, passive HER2-targeted immunotherapy using monoclonal antibodies (mAb, e.g. Trastuzumab and Pertuzumab) has proven to be an effective treatment modality, which has dramatically improved clinical outcomes. Unfortunately, mAb therapy is very expensive and the repeated injections of high doses can be associated with severe side-effects that reduce efficacy. Vaccines are highly cost-effective, but overall progress in development of anti-cancer vaccines based on cancer-associated antigens (e.g. HER2) has been hampered by inherent immune-tolerogenic mechanisms rendering the immune system incapable of reacting against the body’s own cells/proteins (i.e. self-antigens). Consequently, many attempts to develop anti-cancer vaccines have failed in clinical trials due to insufficient immunogenicity. To circumvent this central issue, we have developed a proprietary virus-like particle (VLP)-based vaccine delivery platform. Notably, the VLP-platform is currently the only available technology to effectively facilitate multivalent “virus-like” display of large/complex vaccine antigens. This is key to overcome immune-tolerance and enable induction of therapeutically potent antibody responses directed against cancer-associated self-antigens. In this talk I will discuss the non-viral Drosophila S2 insect cell production system and how it was applied to the production of hHer2/neu antigen, including using advanced production methods such as perfusion for clinical material manufacture. Furthermore, I will present our data from a transgenic mouse model for spontaneous breast cancer development, where high-density display of the HER2 extracellular domain on the surface of virus-like particles (VLPs) enables induction of therapeutically potent anti-HER2 responses. Split-protein tag/catcher conjugation was used to facilitate directional covalent attachment of HER2 to the surface of icosahedral bacteriophage-derived VLPs, thereby harnessing the VLP platform to effectively overcome B-cell tolerance. Vaccine efficacy was demonstrated both in prevention and therapy of mammary carcinomas in HER2 transgenic mice. Thus, the HER2-VLP vaccine shows promise as a new strategy for treatment of HER2-positive cancer. The modular VLP system may also represent an effective tool for development of self-antigen based vaccines against other non-communicable diseases