Angelica Facoetti

Human Leukocyte Antigen and Antigen Processing Machinery Component Defects in Astrocytic Tumors

Purpose: To determine the frequency of abnormalities in human leukocyte antigen (HLA) and antigen processing machinery (APM) component expression in malignant brain tumors. This information may contribute to our understanding of the immune escape mechanisms used by malignant brain tumors because HLA antigens mediate interactions of tumor cells with the hosts immune system. Experimental Design: Eighty-eight surgically removed malignant astrocytic tumors, classified according to the WHO criteria, were stained in immunoperoxidase reactions with monoclonal antibody recognizing monomorphic, locus-specific, and allospecific determinants of HLA class I antigens, β2-microglobulin, APM components (LMP2, LMP7, TAP1, TAP2, calnexin, calreticulin, and tapasin), and HLA class II antigens. Results: HLA class I antigens were lost in ∼50% of the 47 glioblastoma multiforme (GBM) lesions and in ∼20% of the 18 grade 2 astrocytoma lesions stained. Selective HLA-A2 antigen loss was observed in ∼80% of the 24 GBM lesions and in ∼50% of the 12 grade 2 astrocytoma lesions stained. HLA class I antigen loss was significantly (P < 0.025) correlated with tumor grade. Among the APM components investigated, tapasin expression was down-regulated in ∼20% of the GBM lesions analyzed; it was associated, although not significantly, with HLA class I antigen down-regulation and tumor grade. HLA class II antigen expression was detected in ∼30% of the 44 lesions analyzed. Conclusion: The presence of HLA antigen defects in malignant brain tumors may provide an explanation for the relatively poor clinical response rates observed in the majority of the T cell–based immunotherapy clinical trials conducted to date in patients with malignant brain tumors.

Sympathectomy or Doxazosin, But Not Propranolol, Blunt Myocardial Interstitial Fibrosis in Pressure-Overload Hypertrophy

The adaptive changes that develop in the pressure-overloaded left ventricular (LV) myocardium include cardiomyocyte hypertrophy and interstitial fibrosis. Although the former is known to depend to a sizeable extent on sympathetic (over)activity, little information exists whether the same applies to the latter, ie, whether excess catecholamine exposure contributes to the imbalance between collagen deposition by fibroblasts and degradation by matrix metalloproteases (MMPs), eventually leading to LV collagen accumulation. Sprague-Dawley rats were subjected to abdominal aortic banding (B) or sham operation (S) and treated with &bgr;-blockade (Bb, oral propranolol, 40 mg/kg per day), chemical sympathectomy (Sx, 6-hydroxydopamine, 150 mg/kg intraperitoneal twice per week) or vehicle (Vh). Ten weeks later, systolic blood pressure, LV weight, collagen abundance (computer-aided histology), zymographic matrix metalloproteinase (MMP)-2 activity and its specific tissue inhibitor concentration (TIMP-2) were measured. Both sympathectomy and &bgr;-blockade failed to attenuate the banding-induced blood pressure elevation but significantly attenuated the attendant LV hypertrophy. As expected, pressure-overload hypertrophy was associated with interstitial fibrosis (collagen: 4.37±1.23% BVh versus 1.23±0.44% SVh, P<0.05), which was abolished by sympathectomy (2.55±1.31%, P=not significant versus SSx) but left unchanged by &bgr;-blockade (4.11±1.23%, P<0.05 versus both SBb and BSx). &bgr;-blockade, but not sympathectomy, was also associated with an increased TIMP-2/MMP-2 ratio (P<0.05), indicating reduced interstitial collagenolytic activity. In separate groups of banded and sham-operated rats, treatment with the &agr;-receptor blocker doxazosin (10 mg/kg per day) displayed similar antifibrotic and biochemical effects as sympathectomy. Thus in the course of experimental pressure overload, the sympathetic nervous system plays a major pro-fibrotic role, which is mediated via &agr;-adrenergic but not &bgr;-adrenergic receptors.

Biological mechanisms of normal tissue damage: importance for the design of NTCP models.

The normal tissue complication probability (NTCP) models that are currently being proposed for estimation of risk of harm following radiotherapy are mainly based on simplified empirical models, consisting of dose distribution parameters, possibly combined with clinical or other treatment-related factors. These are fitted to data from retrospective or prospective clinical studies. Although these models sometimes provide useful guidance for clinical practice, their predictive power on individuals seems to be limited. This paper examines the radiobiological mechanisms underlying the most important complications induced by radiotherapy, with the aim of identifying the essential parameters and functional relationships needed for effective predictive NTCP models. The clinical features of the complications are identified and reduced as much as possible into component parts. In a second step, experimental and clinical data are considered in order to identify the gross anatomical structures involved, and which dose distributions lead to these complications. Finally, the pathogenic pathways and cellular and more specific anatomical parameters that have to be considered in this pathway are determined. This analysis is carried out for some of the most critical organs and sites in radiotherapy, i.e. spinal cord, lung, rectum, oropharynx and heart. Signs and symptoms of severe late normal tissue complications present a very variable picture in the different organs at risk. Only in rare instances is the entire organ the critical target which elicits the particular complication. Moreover, the biological mechanisms that are involved in the pathogenesis differ between the different complications, even in the same organ. Different mechanisms are likely to be related to different shapes of dose effect relationships and different relationships between dose per fraction, dose rate, and overall treatment time and effects. There is good reason to conclude that each type of late complication after radiotherapy depends on its own specific mechanism which is triggered by the radiation exposure of particular structures or sub-volumes of (or related to) the respective organ at risk. Hence each complication will need the development of an NTCP model designed to accommodate this structure.

A Monte Carlo Study of the Radiation Quality Dependence of DNA Fragmentation Spectra

Abstract We simulated the irradiation of human fibroblasts with γ rays, protons and helium, carbon and iron ions at a fixed dose of 5 Gy. The simulations were performed with the biophysical Monte Carlo code PARTRAC. From the output of the code, containing in particular the genomic positions of the radiation-induced DNA double-strand breaks (DSBs), we obtained the DNA fragmentation spectra. Very small fragments, in particular those related to “complex lesions” (few tens of base pairs), are probably very important for the late cellular consequences, but their detection is not possible with the common experimental techniques. We paid special attention to the differences among the various ions in the production of these very small fragments; in particular, we compared the fragmentation spectra for ions of the same specific energy and for ions of the same LET (linear energy transfer). As found previously for iron ions, we found that the RBE (relative biological effectiveness) for DSB production was considerably higher than 1 for all high-LET radiations considered. This is at variance with the results obtainable from experimental data, and it is due to the ability to count the contribution of small fragments. It should be noted that for a given LET this RBE decreases with increasing ion charge, due mainly to the increasing mean energy of secondary electrons. A precise quantification of the DNA initial damage can be of great importance for both radiation protection, particularly in open-space long-term manned missions, and hadrontherapy.

Effects of ionizing radiation on cell-to-cell communication.

Abstract Cell-to-cell signaling has become a significant issue in radiation biology due to experimental evidence, accumulated primarily since the early 1990s, of radiation-induced bystander effects. Several candidate mediators involved in cell-to-cell communication have been investigated and proposed as being responsible for this phenomenon, but the current investigation techniques (both theoretical and experimental) of the mechanisms involved, due to the particular set-up of each experiment, result in experimental data that often are not directly comparable. In this study, a comprehensive approach was adopted to describe cell-to-cell communication (focusing on cytokine signaling) and its modulation by external agents such as ionizing radiation. The aim was also to provide integrated theoretical instruments and experimental data to help in understanding the peculiarities of in vitro experiments. Theoretical/modeling activities were integrated with experimental measurements by (1) redesigning a cybernetic model (proposed in its original form in the 1950s) to frame cell-to-cell communication processes, (2) implementing and developing a mathematical model, and (3) designing and carrying out experiments to quantify key parameters involved in intercellular signaling (focusing as a pilot study on the release and decay of IL-6 molecules and their modulation by radiation). This formalization provides an interpretative framework for understanding the intercellular signaling and in particular for focusing on the study of cell-to-cell communication in a “step-by-step” approach. Under this model, the complex phenomenon of signal transmission was reduced where possible into independent processes to investigate them separately, providing an evaluation of the role of cell communication to guarantee and maintain the robustness of the in vitro experimental systems against the effects of perturbations.

DNA fragmentation induced in human fibroblasts by 56Fe ions: Experimental data and monte carlo simulations

Abstract Campa, A., Alloni, D., Antonelli, F., Ballarini, F., Belli, M., Dini, V., Esposito, G., Facoetti, A., Friedland, W., Furusawa, Y., Liotta, M., Ottolenghi, A., Paretzke, H. G., Simone, G., Sorrentino, E. and Tabocchini, M. A. DNA Fragmentation Induced in Human Fibroblasts by 56Fe Ions: Experimental Data and Monte Carlo Simulations. Radiat. Res. 171, 438–445 (2009). We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1–5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1–23 kbp and 23–5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1–5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since γ rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

Paraneoplastic brainstem encephalitis in a patient with malignant fibrous histiocytoma and atypical anti-neuronal antibodies

Sirs: Paraneoplastic brainstem encephalitis (BE) is often part of an encephalomyeloneuritis, but may also occur as an isolated clinical entity [4]. It is mostly associated with small cell lung cancer (SCLC), yet some patients develop BE in the course of other tumours. We describe a case of BE associated with a malignant fibrous histiocytoma (MFH) and an atypical anti-neuronal antibody. A 74-year-old man presented with a 2 months history of myoclonus involving the upper extremities. Neurological examination confirmed myoclonus and revealed increased tendon reflexes on the right. Blood studies were normal except for a mildly increased serum level of alfa-fetoprotein. Brain MRI demonstrated only mild cerebral and cerebellar atrophy. The patient was treated with clonazepam with improvement of the myoclonus. The search for antineuronal antibodies in the serum revealed the presence of an atypical antibody, raising the hypothesis of a paraneoplastic neurological disorder (PND). Abdominal CT disclosed a retroperitoneal mass and multiple hepatic and lymphatic formations. Biopsy of a lateral aortic lymph node showed a MFH. At this time, the patient’s neurological state had markedly deteriorated, with dysarthria, truncal and appendicular ataxia, right pyramidal syndrome, left hypoglossal palsy, skew deviation, convergence nystagmus and diplopia with paresis of the right oculomotor nerve. Brain MRI was unchanged. Cerebrospinal fluid (CSF) examination showed mild increase in albumin, normal cell count and intrathecal Ig synthesis with IgG index of 0.77 (normal upper limit for our laboratory < 0.70) and some oligoclonal bands exclusively in the CSF. Testing for anti-neuronal antibodies showed a 1:2000 titre in the serum, consistent with the diagnosis of paraneoplastic BE, and a 1:10 titre in the CSF. The patient underwent surgical removal of the retroperitoneal mass followed by successful chemoand radio-therapy, with oncological remission and neurological improvement. Six months later, no neurological abnormality was present. One year after the diagnosis, he presented again with dysarthria and gait ataxia. CT revealed a retroperitoneal tumour recurrence and an enlarged hepatic metastasis. The patient underwent surgery for excision of the retroperitoneal mass and died from post-operative complications after one week. Serial samples of serum and CSF, obtained during the course of the disease, were examined for the presence of anti-neuronal antibodies. The immunohistochemical analysis using sections of rat’s cerebellum demonstrated an atypical reactivity directed towards axons in the granular and basal molecular layer and around the Purkinje cells (Fig. 1). The reactivity persisted in the serum with unchanged titre after tumour removal. Immunoblot against recombinant Hu-D, Yo and Ri was negative. Western-blot analysis usLETTER TO THE EDITORS

The G‐quadruplex‐stabilizing ligand RHPS4 enhances sensitivity of U251MG glioblastoma cells to clinical carbon ion beams

The pentacyclic acridine RHPS4 is a highly potent and specific G‐quadruplex (G4) ligand, which binds and stabilizes telomeric G4 leading to the block of the replication forks at telomeres and consequently to telomere dysfunctionalization. In turn, the cell recognizes unprotected telomeres as DNA double‐strand breaks with consequent activation of DNA repair response at telomeres, cellular growth impairment, and death. Data from the literature showed the capability of this compound to sensitize U251MG glioblastoma radioresistant cell line to X‐rays sparsely ionizing radiations. In the present paper, it was investigated whether RHPS4 is also able to increase the effect of clinical carbon ion beams (cells irradiated in the middle of a spread‐out Bragg peak, in the energy range of 246–312 MeV·μm−1 and a dose‐averaged linear energy transfer of 46 keV·μm−1). Interestingly, also for charged particles whose damage inflicted to DNA is more complex than that of sparsely ionizing radiations and results in higher Relative Biological Effectiveness (RBE), RHPS4 significantly potentiated the radiation effect in terms of cell killing, delayed rejoining of DNA double‐strand breaks (γ‐H2AX and 53BBP1 immunofluorescence staining), chromosome aberrations (pan‐centromeric/telomeric FISH and multicolor FISH), and G2/M‐phase accumulation in GBM cells. Overall, the results provide the first evidence that the combined administration of the G4‐ligand RHPS4 with charged particles interfere with cellular processes involved in cell survival leading to radiosensitization of highly radioresistant tumor cells.

Morphological analysis of amoeboid-mesenchymal transition plasticity after low and high LET radiation on migrating and invading pancreatic cancer cells

Background/Aim: Cell migration and invasion are fundamental components of tumor cell metastasis that represent the biggest threat to the survival and quality of life of cancer patients. There is clear evidence that ionizing radiation can differently modulate migration and invasiveness of cancer cells depending on the cell lines, the doses and the radiation types investigated. This suggests that motile cells are able to adopt different migration strategies according to their molecular characteristics and external signals. Materials and Methods: In this study, a morphological analysis was performed on pancreatic cancer Aspc-1 cells to evaluate the amoeboid–mesenchymal mobility transition in several experimental conditions considering the role played by factors released by normal and tumor cells, in basal conditions and after low and high Linear Energy Transfer (LET) irradiation. Results and Conclusion: The migratory behavior of Aspc-1 cells is modulated by factors released by normal fibroblasts and tumor cells, and this is in turn modulated by both the radiation dose and the radiation quality.

Uptake of 18F-FET and 18F-FCH in Human Glioblastoma T98G Cell Line after Irradiation with Photons or Carbon Ions

The differential diagnosis between recurrence of gliomas or brain metastases and this phenomenon is important in order to choose the best therapy and predict the prognosis but is still a big problem for physicians. The new emerging MRI, CT, and PET diagnostic modalities still lack sufficient accuracy. Radiolabeled choline and amino acids have been reported to show great tumor specificity. We studied the uptake kinetics of [18F]fluoromethyl-choline (FCH) and O-(2-[18F]fluoroethyl)-L-tyrosine (FET) by the T98G human glioblastoma cells from 20 to 120 min after irradiation either with photons at 2-10-20 Gy or with carbon ions at 2 Gy (at the National Centre for Oncological Hadrontherapy (CNAO), Pavia, Italy). We also evaluated the cell death and morphology changes induced by radiation treatment. Both FET and FCH are able to trace tumor behavior in terms of higher uptake for increased doses of radiation treatment, due to the upregulation of cells attempts to repair nonlethal damage. Our data suggest that both FCH and FET could be useful to analyze the metabolic pathways of glioblastoma cells before and after radiotherapy. Physicians will have to consider the different kinetics pathways of uptake concerning the two radiopharmaceuticals.