Arianna Citti

Tumor-infiltrating T lymphocytes improve clinical outcome of therapy-resistant neuroblastoma

Neuroblastoma grows within an intricate network of different cell types including epithelial, stromal and immune cells. The presence of tumor-infiltrating T cells is considered an important prognostic indicator in many cancers, but the role of these cells in neuroblastoma remains to be elucidated. Herein, we examined the relationship between the type, density and organization of infiltrating T cells and clinical outcome within a large collection of neuroblastoma samples by quantitative analysis of immunohistochemical staining. We found that infiltrating T cells have a prognostic value greater than, and independent of, the criteria currently used to stage neuroblastoma. A variable in situ structural organization and different concurrent infiltration of T-cell subsets were detected in tumors with various outcomes. Low-risk neuroblastomas were characterized by a higher number of proliferating T cells and a more structured T-cell organization, which was gradually lost in tumors with poor prognosis. We defined an immunoscore based on the presence of CD3+, CD4+ and CD8+ infiltrating T cells that associates with favorable clinical outcome in MYCN-amplified tumors, improving patient survival when combined with the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) status. These findings support the hypothesis that infiltrating T cells influence the behavior of neuroblastoma and might be of clinical importance for the treatment of patients.

IRF1 and NF-kB Restore MHC Class I-Restricted Tumor Antigen Processing and Presentation to Cytotoxic T Cells in Aggressive Neuroblastoma

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.

Clinical and ultrastructural spectrum of diffuse lung disease associated with surfactant protein C mutations

Genetic defects of surfactant metabolism are associated with a broad range of clinical manifestations, from neonatal respiratory distress syndrome to adult interstitial lung disease. Early therapies may improve symptoms but diagnosis is often delayed owing to phenotype and genotype variability. Our objective was to characterize the cellular/ultrastructural correlates of surfactant protein C (SP-C) mutations in children with idiopathic diffuse lung diseases. We sequenced SFTPC – the gene encoding SP-C – SFTPB and ABCA3, and analyzed morphology, ultrastructure and SP expression in lung tissue when available. We identified eight subjects who were heterozygous for SP-C mutations. Median age at onset and clinical course were variable. None of the mutations were located in the mature peptide-encoding region, but were either in the pro-protein BRICHOS or linker C-terminal domains. Although lung morphology was similar to other genetic surfactant metabolism disorders, electron microscopy studies showed specific anomalies, suggesting surfactant homeostasis disruption, plus trafficking defects in the four subjects with linker domain mutation and protein misfolding in the single BRICHOS mutation carrier in whom material was available. Immunolabeling studies showed increased proSP-C staining in all cases. In two cases, amyloid deposits could be identified. Immunochemistry and ultrastructural studies may be useful for diagnostic purposes and for genotype interpretation.

Ultrastructural Characterization of Genetic Diffuse Lung Diseases in Infants and Children: A Cohort Study and Review

Abstract Pediatric diffuse lung diseases are rare disorders with an onset in the neonatal period or in infancy, characterized by chronic respiratory symptoms and diffuse interstitial changes on imaging studies. Genetic disorders of surfactant homeostasis represent the main etiology. Surfactant protein B and ABCA3 deficiencies typically cause neonatal respiratory failure, which is often lethal within a few weeks or months. Although heterozygous ABCA3 mutation carriers are mostly asymptomatic, there is growing evidence that monoallelic mutations may affect surfactant homeostasis. Surfactant protein C mutations are dominant or sporadic disorders leading to a broad spectrum of manifestations from neonatal respiratory distress syndrome to adult pulmonary fibrosis. The authors performed pathology and ultrastructural studies in 12 infants who underwent clinical lung biopsy. One carried a heterozygous SP-B mutation, 3 carried SP-C mutations, and 7 carried ABCA3 mutations (5 biallelic and 2 monoallelic). Optical microscopy made it possible to distinguish between surfactant-related disorders and other forms. One of the ABCA3 monoallelic carriers had morphological features of alveolar capillary dysplasia, a genetic disorder of lung alveolar, and vascular development. One patient showed no surfactant-related anomalies but had pulmonary interstitial glycogenosis, a developmental disorder of unknown origin. Electron microscopy revealed specific lamellar bodies anomalies in all SP-B, SP-C, and ABCA3 deficiency cases. In addition, the authors showed that heterozygous ABCA3 mutation carriers have an intermediate ultrastructural phenotype between homozygous carriers and normal subjects. Lung biopsy is an essential diagnostic procedure in unexplained diffuse lung disorders, and electron microscopy should be performed systematically, since it may reveal specific alterations in genetic disorders of surfactant homeostasis.

Expression of multidrug resistance-associated proteins in paediatric soft tissue sarcomas before and after chemotherapy

Expression of multidrug resistance (MDR) proteins is thought to significantly contribute to the different biological/clinical behaviour of soft tissue sarcomas (STS) of various histological types and clinicopathological stages, as they are responsible for active efflux of cytotoxic drugs from tumour cells. We investigated the expression of 3 MDR proteins, i.e., permeability glycoprotein 1 (P-gp), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance 3 (MDR3), in 43 STS specimens from newly-diagnosed paediatric patients, 31 with rhabdomyosarcoma (RMS) and 12 with non-RMS STS. To assess the influence of chemotherapy on STS drug resistance, the number of MDR-associated protein-positive cells was determined in 15 patients on both primary lesions before chemotherapy and on residual tumour after chemotherapy. At least one of the MDR-associated proteins tested was detected in 84% of primary untreated STS specimens. In these specimens, MRP1 was detected in a high percentage (70%) of the cases, followed by MDR3 in 58% and P-gp in 44%. Many specimens showed co-expression of two different MDR proteins. Interestingly, MDR3 was significantly associated with the presence of PAX3/PAX7-FKHR transcripts in RMS (p<0.05). Moreover, expression of MRP1 and MDR3 was significantly more frequent in group III and IV tumours as compared with those of groups I and II (p<0.01). After chemotherapy MRP1, MDR3 and, to a lesser extent, P-gp expression was found to be increased in most of the samples. The frequent expression of these MDR-associated proteins in primary tumour cells before chemotherapy and the increase of their levels after chemotherapy, suggest that these proteins play a pivotal role in conferring drug resistance and in producing therapy-induced differentiation on STS.

Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

High-Resolution Array CGH Profiling Identifies Na/K Transporting ATPase Interacting 2 (NKAIN2) as a Predisposing Candidate Gene in Neuroblastoma

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.