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Introduction: B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug conjugate (ADC) directed against the B7-H3 antigen. It is composed of an anti-B7-H3 humanized IgG1/kappa monoclonal antibody chemically conjugated through a cleavable valine-citrulline linker to a du ocarmycin-hydroxy b enzamide a zaindole (vc-seco-DUBA). Vobra duo has shown preliminary clinical activity in B7-H3-expressing tumors., Methods: B7-H3 expression was evaluated by flow-cytometry in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by the water-soluble tetrazolium salt,MTS, proliferation assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by annexin V staining. Orthotopic, pseudometastatic, and resected mouse NB models were developed to mimic disease conditions related to primary tumor growth, metastases, and circulating tumor cells with minimal residual disease, respectively., Results: All human NB cell lines expressed cell surface B7-H3 in a unimodal fashion. Vobra duo was cytotoxic in a dose-dependent and time-dependent manner against all cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). Vobra duo was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of vobra duo when co-cultured with human B7-H3-expressing cells, demonstrating bystander activity. In orthotopic and pseudometastatic mouse models, weekly intravenous treatments with 1 mg/kg vobra duo for 3 weeks delayed tumor growth compared with animals treated with an irrelevant (anti-CD20) duocarmycin-ADC. Vobra duo treatment for 4 weeks further increased survival in both orthotopic and resected NB models. Vobra duo compared favorably to TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for NB relapsed disease, with tumor relapse delayed or arrested by two or three repeated 4-week vobra duo treatments, respectively. Further increased survival was observed in mice treated with vobra duo in combination with TOTEM. Vobra duo treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities., Conclusion: Vobra duo exerts relevant antitumor activity in preclinical B7-H3-expressing NB models and represents a potential candidate for clinical translation., Competing Interests: Competing interests: EB and DL are employees of MacroGenics and receive salary and stocks as part of their compensations. The other authors do not have competing interests to declare., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Neuroblastoma is a biologically heterogeneous extracranial tumor, derived from the sympathetic nervous system, that affects most often the pediatric population. Therapeutic strategies relying on aggressive chemotherapy, surgery, radiotherapy, and immunotherapy have a negative outcome in advanced or recurrent disease. Here, spherical polymeric nanomedicines (SPN) are engineered to co-deliver a potent combination therapy, including the cytotoxic docetaxel (DTXL) and the natural wide-spectrum anti-inflammatory curcumin (CURC). Using an oil-in-water emulsion/solvent evaporation technique, four SPN configurations were engineered depending on the therapeutic payload and characterized for their physico-chemical and pharmacological properties. All SPN configurations presented a hydrodynamic diameter of ∼ 185 nm with a narrow size distribution. A biphasic release profile was observed for all the configurations, with almost 90 % of the total drug mass released within the first 24 h. SPN cytotoxic potential was assessed on a panel of human neuroblastoma cells, returning IC 50 values in the order of 1 nM at 72 h and documenting a strong synergism between CURC and DTXL. Therapeutic efficacy was tested in a clinically relevant orthotopic model of neuroblastoma, following the injection of SH-SY5Y-Luc + cells in the left adrenal gland of athymic mice. Although ∼ 2 % of the injected SPN per mass tissue reached the tumor, the overall survival of mice treated with CURC/DTXL-SPN was extended by 50 % and 25 % as compared to the untreated control and the monotherapies, respectively. In conclusion, these results demonstrate that the therapeutic potential of the DTXL/CURC combination can be fully exploited only by reformulating these two compounds into systemically injectable nanoparticles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

Caelyx and Myocet are clinically used liposomal forms of doxorubicin (Dox). To explore ways to improve their therapeutic index, we have studied their activity in vitro and in vivo when locally delivered by fibrin gels (FBGs). In vivo local toxic and anti-tumour activities of loaded FBGs were assessed in two immunodeficient mouse orthotopic human neuroblastoma (NB) models after application in the visceral space above the adrenal gland, either still tumour-bearing or after tumour removal. In parallel, in vitro assays were used to mimic the in vivo overlaying of FBGs on the tumour surface. FBGs were prepared with different concentrations of fibrinogen (FG) and clotted in the presence of Ca 2+ and thrombin. The in vitro assays showed that FBGs loaded with Myocet possess a cytotoxic activity against NB cell lines generally greater than those loaded with free Dox or Caelyx. In vivo FBGs loaded with Myocet showed lower general and local toxicities as compared to gels loaded with Caelyx or free Dox, and also to free Dox administered i.v. (all treatments with Dox at 2.5 mg/Kg). The anti-tumour activity, evaluated in the two mouse orthotopic NB models of adjuvant and neo-adjuvant therapy, resulted in a better performance of FBGs loaded with Myocet compared to the other local (FBGs loaded with Caelyx or free Dox) or systemic (free Dox) treatments (administered at 2.5 and 5 mg/Kg Dox). Specifically, the application of FBGs at 40 mg/mL in the adjuvant model caused 92 % tumour volume reduction, while by the neo-adjuvant application of FBGs at 22 mg/mL a re-growing tumour volume reduction of 89 % was obtained. Taken together, our in vitro and in vivo results indicate a significantly higher activity for the FBGs loaded with Myocet. In particular, the lower toicity coupled with the higher anti-tumour activity on both the local treatment modalities strongly suggest a better therapeutic index when Myocet is administered through FBGs. Therefore, FBGs loaded with Myocet may be considered as a possible new tool for the loco-regional treatment of NB or even other tumour histotypes treatable by loco-regional chemotherapy., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino β-cyclodextrin polymer (oCD-NH 2 /Dox). Here we extend these studies by in vitro and in vivo evaluations., Methods: An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc + cells implanted into the left adrenal gland)., Results: In vitro data show that FBGs loaded with oCD-NH 2 /Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca 2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH 2 /Dox than those loaded with Dox., Conclusion: Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH 2 /Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or adipose tissue can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural scaffold of MSCs, to deliver PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with a long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, DMFAT loaded with PTX prevents or delays NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may represent very innovative natural biomaterials able to localize and release anti-cancer molecules at the tumor site, helping to fight cancer in human., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Neuroblastoma (NB) is an embryonal tumor originating from undifferentiated neural crest cell, highly heterogeneous ranging from spontaneous regression to progression despite multimodal treatments. Approximately, 20% of patients are refractory to frontline therapy and 50% will relapse/progress after an initial response. The overall five year survival for high-risk neuroblastoma ranges from 35-45%. Despite enhanced understanding of NB biology and the addition of myeloablative chemotherapy, isotretinoin and immunotherapy, survival for high risk NB remains less than 50%. Areas covered: This review summarizes and gives a critical overview of phase II trials investigating therapies for relapsed-refractory and high risk neuroblastoma. Expert opinion: Several novel molecules have been developed and are currently under investigation for the treatment of NB. The trend of novel targeted agents is one towards individualized, tailored therapy, based on the molecular and biological differences that characterize tumors that seem similar based solely on histological analysis. The task of developing new molecules is particularly difficult for NB, given the recurrent development of new patterns of drug resistance. However, even if current research is focused towards identifying the best treatments for each children and young adult with a NB defined disease, a deeper knowledge of the molecular biology and genetics is needed.

The skin is a possible site of extramedullary localization in multiple myeloma (MM) patients; however, the mechanisms involved in this process are poorly understood. We describe the case of a refractory MM patient who developed a cutaneous localization under bortezomib treatment and we further expanded observations in other eight MM patients. We focused on the expression of genes involved in plasma cell skin homing, including CCR10, which was highly expressed. Moreover, we observed a lack of CXCR4 surface expression and the down-regulation of ICAM1/CD54 throughout the progression of the disease, suggesting a possible mechanism driving the escape of MM cells from the bone marrow into the skin.

The bromodomain inhibitor OTX015 (MK-8628) has shown anti-lymphoma activity as a single agent in both the preclinical and clinical settings, as well as in vitro synergism with several anticancer agents. Here, we report in vivo data for OTX015 in combination with the histone deacetylase inhibitor vorinostat, the Bruton's tyrosine kinase inhibitor ibrutinib, the anti-CD20 monoclonal antibody rituximab, and the mTOR inhibitor everolimus in a diffuse large B cell lymphoma model. The antitumor effect of OTX015-containing combinations in SU-DHL-2 xenografts in mice was much stronger than the activity of the corresponding single agents with almost complete tumor eradication for all four combinations. Pharmacokinetic analyses showed similar OTX015 levels in plasma and tumor samples of approximately 1.5 μM, which is equivalent to the concentration showing strong in vitro activity. For all four combinations, mean terminal levels of the bromodomain inhibitor differed from those in mice exposed to single agent OTX015, indicating a need for thorough pharmacokinetic investigations in phase I combination studies. In conclusion, our results provide a strong rationale to explore OTX015-containing combinations in the clinical lymphoma setting., Competing Interests: FB, KR, AS and EZ have received institutional research funds from Oncology Therapeutic Development. Esteban Cvitkovic was founder and CSO of Oncoethix SA, and CEO of Oncology Therapeutic Development. Maria E. Riveiro was an employee of Oncology Therapeutic Development. EZ has received research funds from Celgene, Novartis, Mundipharma, Roche, Pharmacyclics Inc., Johnson & Johnson's Janssen Pharmaceutical, Gilead. The remaining authors have no conflicts of interest.

Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors.

Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-containing proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015., Experimental Design: We assessed the preclinical activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo experiments to identify the mechanism of action and the genetic features associated with sensitivity to the compound., Results: OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo experiments showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, especially with mTOR and BTK inhibitors. Gene expression signatures associated with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compound induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell-like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11., Conclusions: Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clinical investigation of OTX015 as single agent and in combination therapies., (©2015 American Association for Cancer Research.)

Neuroblastoma (NB), the most common and deadly extracranial solid tumour of childhood, represents a challenging in paediatric oncology. Soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) is a cancer cell-specific molecule exerting remarkable anti-tumour activities against paediatric malignancies both in vitro and in preclinical settings. However, due to its too fast elimination and to the undesired related side effects, the improvement of sTRAIL in vivo bioavailability and the specific delivery to the tumour is mandatory for increasing its therapeutic efficacy. In this manuscript, we developed an innovative pegylated liposomal formulation carrying the sTRAIL at the outer surface (sTRAIL-SL) with the intent to improve its serum half-life and increase its efficacy in vivo, while reducing side effects. Furthermore, the possibility to combine sTRAIL-SL with the proteasome inhibitor Bortezomib (BTZ) was investigated, being BTZ able to sensitize tumour cells toward TRAIL-induced apoptosis. We demonstrated that sTRAIL preserved and improved its anti-tumour activity when coupled to nanocarriers. Moreover, sTRAIL-SL ameliorated its PK profile in blood allowing sTRAIL to exert a more potent anti-tumour activity, which led, upon BTZ priming, to a statistically significant enhanced life spans in two models of sTRAIL-resistant NB-bearing mice. Finally, mechanistic studies indicated that the combination of sTRAIL with BTZ sensitized sTRAIL-resistant NB tumour cells to sTRAIL-induced cell death, both in vitro and in vivo, through the Akt/GSK3/β-catenin axis-dependent mechanism. In conclusion, our results suggest that sTRAIL-SL might be an efficient vehicle for sTRAIL delivery and that its use in clinic, in combination with BTZ, might represent an adjuvant strategy for the treatment of stage IV, sTRAIL-resistant, NB patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: High levels of cyclin E (CCNE) are accompanied by shorter survival in cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (CHOP)-treated diffuse large B-cell lymphomas (DLBCL), independent of the international prognostic index (IPI). Data on the prognostic role of CCNE in the 'rituximab (R)-era' are lacking., Methods: To test reproducibility and applicability of observations from the 'pre-R era' to the 'R era', we examined the prognostic role of CCNE expression by immunohistochemistry in 1579 DLBCL on tissue microarrays (TMA); 339 patients were treated by CHOP and 635 by R-CHOP., Results: 1209 samples (77%) were evaluable; failures were due to missing TMA punches and fixation artefacts. Mean expression of CCNE was 13% (0-85%); applying a cut-off of >16%, 382 DLBCL (31%) were positive. CCNE did not correlate with any of the known variables (IPI, primary site, cell of origin, proliferation, and BCL2- or C-MYC rearrangements). We were able to reproduce data suggesting an IPI- and response to therapy independent, negative prognostic impact of CCNE in CHOP-treated DLBCL patients: CCNE-positive cases had a median survival of 16 months compared with 57 months in negative ones (p=0.012). In R-CHOP-treated patients the prognostic impact of CCNE was abrogated and only IPI, cell of origin and response to therapy had a prognostic significance., Conclusions: Addition of R to CHOP overcomes the negative prognostic impact of CCNE in DLBCL. Thus, R not only prolongs survival in DLBCL but also serves a cautionary note that prognostic factors should not be transferred into the 'R era' without proper scientific studies.

Neuroblastoma is an embryonal tumor originating from the simpatico-adrenal lineage of the neural crest. It approximately accounts for about 15% of all pediatric oncology deaths. Despite advances in multimodal therapy, metastatic neuroblastoma tumors at diagnosis remain a clinical challenge. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of neuroblastoma cells. Among them, fenretinide (HPR) has been considered one of the most promising anti-tumor agent but it is partially efficacious due to both poor aqueous solubility and rapid metabolism. Here, we have developed a novel HPR formulation, by which the drug was encapsulated into sterically stabilized nanoliposomes (NL[HPR]) according to the Reverse Phase Evaporation method. This procedure led to a higher structural integrity of liposomes in organic fluids for a longer period of time, in comparison with our previous liposomal formulation developed by the film method. Moreover, NL[HPR] were further coupled with NGR peptides for targeting the tumor endothelial cell marker, aminopeptidase N (NGR-NL[HPR]). Orthotopically xenografted neuroblastoma-bearing mice treated with NGR-NL[HPR] lived statistically longer than mice untreated or treated with free HPR (NGR-NL[HPR] vs both control and HPR: P<0.0001). Also, NL[HPR] resulted in a statistically improved survival (NL[HPR] vs both control and HPR: P<0.001) but to a less extent if compared with that obtained with NGR-NL[HPR] (NGR-NL[HPR] vs NL[HPR]: P<0.01). Staining of tumor sections with antibodies specific for neuroblastoma and for either pericytes or endothelial cells evidenced that HPR reduced neuroblastoma growth through both anti-tumor and anti-angiogenic effects, mainly when delivered by NGR-NL[HPR]. Indeed, in this group of mice a marked reduction of tumor progression, of intra-tumoral vessel counts and VEGF expression, together with a marked down-modulation of matrix metalloproteinases MMP2 and MMP9, was observed. In conclusion, the use of this novel targeted delivery system for the apoptotic and antiangiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of neuroblastoma patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)

HS1 (hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL). The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the tyrosine kinase inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EμTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.

Purpose: Treatment of neuroblastoma is successful in less than half of patients with high-risk disease. The antitumor activity of a water soluble pegylated SN38 drug conjugate, EZN-2208, was compared with CPT-11 (a prodrug for SN38) in preclinical models of human neuroblastoma., Experimental Design: The in vitro cytotoxicity of EZN-2208 was tested by counting trypan blue dye- and Annexin V-positive cells, whereas its therapeutic efficacy was evaluated, in terms of survival, and antitumor and antiangiogenic activities, in s.c. luciferase-transfected, pseudometastatic, and orthotopic neuroblastoma animal models., Results: EZN-2208 was about 100-fold more potent than CPT-11 in vitro, by inducing apoptosis/necrosis and p53 expression and by reducing hypoxia-inducible factor (HIF)-1α/HIF-2α expression. EZN-2208 gave superior antitumor effects compared with CPT-11 in neuroblastoma xenografts. EZN-2208 treatment always resulted in lack of tumor detection at the end of trials whereas only small therapeutic effects were observed with CPT-11, as assessed by luciferase assay or tumor size, or even by staining histologic sections of tumors with antibodies recognizing neuroblastoma cells and cell proliferation. In a neuroblastoma model resistant to doxorubicin, cisplatin, vincristine, fenretinide, and topotecan, EZN-2208 induced 100% curability. It also blocked tumor relapse after topotecan-vincristine-doxorubicin combined treatment. Mechanistic experiments showed statistically significantly enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Histone H2ax staining as well as decreased vascular endothelial growth factor, CD31, matrix metalloproteinase (MMP)-2, and MMP-9 expression in tumors removed from EZN-2208-treated mice and radiating vessels invading the tumor implanted onto the chorioallantoic membranes., Conclusions: EZN-2208 should be considered a most promising novel antineuroblastoma agent. An ongoing phase I study in pediatric patients should identify the optimal dose for a phase II study., (©2010 AACR.)

Purpose: Multiple myeloma (MM) derives from plasmablast/plasma cells that accumulate in the bone marrow. Different microenvironmental factors may promote metastatic dissemination especially to the skeleton, causing bone destruction. The balance between osteoclast and osteoblast activity represents a critical issue in bone remodeling. Thus, we investigated whether interluekin-27 (IL-27) may function as an antitumor agent by acting directly on MM cells and/or on osteoclasts/osteoblasts., Experimental Design: The IL-27 direct antitumor activity on MM cells was investigated in terms of angiogenesis, proliferation, apoptosis, and chemotaxis. The IL-27 activity on osteoclast/osteoblast differentiation and function was also tested. In vivo studies were done using severe combined immunodeficient/nonobese diabetic mice injected with MM cell lines. Tumors from IL-27- and PBS-treated mice were analyzed by immunohistochemistry and PCR array., Results: We showed that IL-27 (a) strongly inhibited tumor growth of primary MM cells and MM cell lines through inhibition of angiogenesis, (b) inhibited osteoclast differentiation and activity and induced osteoblast proliferation, and (c) damped in vivo tumorigenicity of human MM cell lines through inhibition of angiogenesis., Conclusions: These findings show that IL-27 may represent a novel therapeutic agent capable of inhibiting directly MM cell growth as well as osteoclast differentiation and activity.

Purpose: To describe the magnetic resonance (MR) imaging and diffusion-weighted (DW) imaging features of ocular adnexal lymphomas (OALs), to determine the diagnostic accuracy of apparent diffusion coefficient (ADC) for discriminating OALs from other orbital mass lesions, and to assess whether variations in ADC constitute a reliable biomarker of OAL response to therapy., Materials and Methods: Institutional ethical committee approval and informed consent were obtained. In this prospective study, 114 white subjects (65 females and 49 males) were enrolled. Thirty-eight patients with histopathologically proved OAL underwent serial MR and DW imaging examination of the orbits. ADCs of OALs were compared with those of normal orbital structures, obtained in 18 healthy volunteers, and other orbital mass lesions, prospectively acquired in 58 patients (20 primary non-OAL neoplasms, 15 vascular benign lesions, 12 inflammatory lesions, 11 metastases). Interval change in ADC of OALs before and after treatment was analyzed in 29 patients. Analysis of covariance and a paired t test were used for statistical analysis., Results: Baseline ADCs in OALs were lower than those in normal structures and other orbital diseases (P < .001). An ADC threshold of 775 x 10(-6) mm(2)/sec resulted in 96% sensitivity, 93% specificity, 88% positive predictive value, 98.2% negative predictive value, and 94.4% accuracy in OAL diagnosis. Following appropriate treatment, 10 (34%) of 29 patients showed OAL volumetric reduction, accompanied (n = 7) or preceded (n = 3) by an increase in ADC (P = .005). Conversely, a further reduction of ADC was observed in the seven patients who experienced disease progression (P < .05)., Conclusion: ADC permits accurate diagnosis of OALs. Interval change in ADC after therapy represents a helpful tool for predicting therapeutic response.

Background: Sharing the common neuroectodermal origin, melanoma and neuroblastoma are tumors widely diffused among adult and children, respectively. Clinical prognosis of aggressive neuroectodermal cancers remains dismal, therefore the search for novel therapies against such tumors is warranted. Curcumin is a phytochemical compound widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Recently, we have synthesized and tested in vitro various curcumin-related compounds in order to select new anti-tumor agents displaying stronger and selective growth inhibition activity on neuroectodermal tumors., Results: In this work, we have demonstrated that the new alpha,beta-unsaturated ketone D6 was more effective in inhibiting tumor cells growth when compared to curcumin. Normal fibroblasts proliferation was not affected by this treatment. Clonogenic assay showed a significant dose-dependent reduction in both melanoma and neuroblastoma colony formation only after D6 treatment. TUNEL assay, Annexin-V staining, caspases activation and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. These apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous melanoma and orthotopic neuroblastoma xenograft models. D6 treated mice exhibited significantly reduced tumor growth compared to both control and curcumin treated ones (Melanoma: D6 vs control: P < 0.001 and D6 vs curcumin P < 0.01; Neuroblastoma: D6 vs both control and curcumin: P < 0.001)., Conclusions: Our data indicate D6 as a good candidate to develop new therapies against neural crest-derived tumors.

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.

Disseminated neuroectoderma-derived tumors, mainly neuroblastoma in childhood and melanoma in the adulthood, are refractory to most current therapeutic regimens and hence the prognosis remains very poor. Preclinical research studies have indicated several agents that show promising therapeutic potential for these neoplasms. However, there appears to be a limitation to their in vivo applicability, mainly due to unfavorable pharmacokinetic properties that lead to insufficient drug delivery to the tumor or metastatic sites or to high systemic or organ-specific toxicity. In this scenario, the focus is on targeted cancer therapy. Encapsulating anticancer drugs in liposomes enables targeted drug delivery to tumor tissue and prevents damage to the normal surrounding tissue. Indeed, sterically stabilized liposomes have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. The identification of tumor-associated antigens and/or genes and the relative ease of manipulating the physicochemical features of liposome hold promise for the development of novel therapeutic strategies that selectively target tumor cells. Combined targeting is still investigated, especially the availability to simultaneously target and kill both the cancer cells and the tumor vasculature. Animal models make it possible to link molecular genetics and biochemistry information to the physiological basis of disease and are important predictive tools that offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutics approaches. Relevant experimental models of human neuroblastoma and melanoma, which better reflect the tumor behavior in patients, are required to evaluate the effectiveness of the various targeted liposomal formulations and their possible systemic and organ-specific toxicity. The most multifunctional targeted liposomes are herein described, with primary attention on testing their efficacy in clinically relevant animal models for the treatment of neuroblastoma and melanoma.

The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. The use of liposomes as drug delivery vehicles for antitumour therapeutics has great potential to revolutionise the future of cancer therapy. As tumour architecture causes liposomes to preferentially accumulate at the tumour site, their use as drug carriers results in the localization of a greater amount of the loaded drug at the tumour site, thus improving cancer therapy and reducing the harmful non-specific side effects of chemotherapeutics. In addition, targeting of liposomal anticancer drugs to antigens expressed or over-expressed on tumour cells provides a very efficient system for increasing the therapeutic indices of the drugs. Animal models allow detailed examination of molecular and physiological basis of diseases and offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutic approaches. Until recently, the most resorted experimental model of paediatric Neuroblastoma (NB) tumour is the subcutaneous xenograft in nude mice. However, the main disadvantage of this animal model is that it does not reflect the metastatic potential of NB cells, ultimately responsible for poor patient survival. A more realistic view of the clinical potential of targeted therapies could be obtained if a tumour model were available that better reflects the growth of advanced NB in children (i.e. large adrenal gland tumours and multiple small metastatic lesions). All current data support this concept and recommend that orthotopic implantation of tumour cells in recipient animals is mandatory for studies of tumour progression, angiogenesis, invasion, and metastasis. This review will focus on the description of the most clinically relevant animal models established to test the efficacy of targeted liposomal anti-tumour formulations for the treatment of Neuroblastoma.

Background: An association between ocular adnexal MALT lymphoma (OAL) and Chlamydia psittaci (Cp) infection has been proposed, and recent reports suggest that doxycycline treatment causes tumor regression in patients with Cp-related OAL. The effectiveness of doxycycline treatment in Cp-negative OAL has not been tested., Methods: In a prospective trial, 27 OAL patients (15 newly diagnosed and 12 having experienced relapse) were given a 3-week course of doxycycline therapy. Objective lymphoma response was assessed by computerized tomography scans or magnetic resonance imaging at 1, 3, and 6 months after the conclusion of therapy and every 6 months during follow-up. Cp infection in patients was determined by touchdown enzyme time-release polymerase chain reaction (TETR-PCR). Statistical tests were two-sided., Results: Eleven patients were Cp DNA-positive and 16 were Cp DNA negative. Doxycycline was well tolerated. At a median follow-up of 14 months, lymphoma regression was complete in six patients, and a partial response (> or = 50% reduction of all measurable lesions) was observed in seven patients (overall response rate [complete and partial responses] = 48%). Lymphoma regression was observed in both Cp DNA-positive patients (seven of 11 experienced regression) and Cp DNA-negative patients (six of 16 experienced regression) (64% versus 38%; P = .25, Fisher's exact test). The three patients with regional lymphadenopathies and three of the five patients with bilateral disease achieved objective response. In relapsed patients, response was observed both in previously irradiated and nonirradiated patients. The 2-year failure-free survival rate among the doxycycline-treated patients was 66% (95% confidence interval = 54 to 78), and 20 of the 27 patients were progression free., Conclusions: Doxycycline is a fast, safe, and active therapy for Cp DNA-positive OAL that was effective even in patients with multiple failures involving previously irradiated areas or regional lymphadenopathies. The responses observed in PCR-negative OAL may suggest a need for development of more sensitive methods for Cp detection and investigation of the potential role of other doxycycline-sensitive bacteria.

Background: Bortezomib is a selective and reversible inhibitor of the 26S proteasome that shows potent antitumor activity in vitro and in vivo against several human cancers of adulthood. No data are available on bortezomib activity against human pediatric neuroblastoma., Methods: Ten neuroblastoma cell lines and suspensions of primary neuroblastoma cells from three patients were tested for sensitivity to bortezomib. Colony formation, cell proliferation, cell cycle progression, and apoptosis were evaluated by a clonogenic assay and by measuring 3H-thymidine incorporation, bromodeoxyuridine uptake, DNA fragmentation, and phosphatidylserine exposure and propidium iodide staining, respectively. Angiogenesis was assessed by the chick embryo chorioallantoic membrane (CAM) assay. Two mouse xenograft models that mimic the growth and spread of neuroblastoma in humans were used to examine in vivo sensitivity of neuroblastoma to bortezomib. All statistical tests were two-sided., Results: Bortezomib inhibited proliferation and colony formation of neuroblastoma cell lines in a time- and dose-dependent manner. The mean bortezomib concentration that caused 50% inhibition of growth was 6.1 nM (95% confidence interval [CI] = 0.9 to 11.3 nM) at 72 hours. Bortezomib-treated neuroblastoma cells were arrested at G2/M and underwent apoptosis (mean percentage of apoptotic cells in four neuroblastoma cell lines treated with 20 nM bortezomib for 24 hours ranged from 20% to 35%, and caspases were activated by two- to fivefold with respect to untreated cells). Similar results were obtained for primary neuroblastoma cells exposed to bortezomib. Bortezomib inhibited angiogenesis in CAMs stimulated by conditioned medium from neuroblastoma cell lines, by neuroblastoma xenografts, and by primary neuroblastoma biopsy specimens (microvessel area: 2.9 x 10(-2) mm2, 95% CI = 1.8 x 10(-2) to 3.8 x 10(-2) mm2 in CAMs treated with biopsy specimens alone and 1.3 x 10(-2) mm2, 95% CI = 1 x 10(-2) to 1.5 x 10(-2) mm2 in CAMs treated with biopsy specimens plus bortezomib, P = .024). In both mouse models, mice treated with bortezomib lived statistically significantly longer than control mice (mean survival time in the pseudometastatic model: 74.2 versus 50.3 days, P<.001; mean survival time in the orthotopic model: 72.3 versus 50.6 days, P<.001)., Conclusions: Bortezomib is an effective inhibitor of neuroblastoma cell growth and angiogenesis. These findings provide the rationale for further clinical investigation of bortezomib in pediatric neuroblastoma.

Tumor homing peptides containing the NGR motif, such as CNGRC and GNGRG, have been used for delivering cytokines, chemotherapeutic drugs, apoptotic peptides, and liposomes to a CD13 isoform expressed in tumor blood vessels. In view of the potential clinical applications of these drugs and considering the risk that NGR peptides could elicit blocking antibodies we have investigated the immunogenic properties of CNGRC and GNGRG in mice and rabbits, using various products containing these residues and different administration schedules. The results suggest that the immunogenicity of the NGR motif is very low, even when it is conjugated to tumor necrosis factor-alpha or to highly immunogenic carrier proteins. Molecular dynamics simulation experiments showed that both peptides have a strong propensity to populate a turn conformation. Superposition of predicted structures to the CTGNGRGEWKC loop of the 5th type I repeat of human fibronectin, a protein that contains four NGR motives, showed that the root mean square deviation of backbones was 0.7A for GNGRG and 0.5A for NGR. These results suggest that NGR peptides could mimic from an immunological point of view a "self" structure, likely the GNGRG loop of fibronectin, with important implications for the use of these targeting peptides in patients.

Purpose: Preliminary results using rituximab in extranodal marginal zone (MALT) non-Hodgkin's lymphoma (NHL) patients seem to indicate a relevant clinical activity. Aim of the present study is to investigate the efficacy of conventional weekly treatment using rituximab in gastric MALT NHL patients resistant/refractory or not suitable for eradication treatment, and to evaluate the relevance of the t(11; 18)(q21; q21) translocation and its possible role as a predictive criteria of response., Patients and Methods: Twenty-seven patients presenting with gastric MALT NHL at any stage, relapsed/refractory to initial treatment or not suitable for eradication were treated with rituximab in a weekly conventional schedule and evaluated for response and relapse. Flourescence in situ hybridization (FISH) analysis for the presence of 18q21 translocation was performed in 21 patients and was evaluated with clinical outcome., Results: Among the 26 evaluated patients, 20 (77%) achieved an objective response. Twelve patients (46%) had a pathological and clinical complete remission, and eight (31%) had a partial response. With a median follow-up of 33 months, only two patients relapsed at 26 and 14 months, respectively. No correlation was founded between FISH analysis and response or relapse., Conclusion: Our experience seems to confirm the clinical activity of rituximab in gastric MALT NHL patients resistant/refractory to antibiotics treatment or not presenting with clinical evidence of Helicobacter pylori infection. The t(11; 18)(q21; q21) translocation seems not to be a predictive marker to response or to subsequent relapse.

Promising new antiangiogenic strategies are emerging for the treatment of cancer and the inhibition of angiogenesis could represent a powerful adjunct to traditional therapy of malignant tumors. Over the last ten years several reports have been published concerning the relationship between tumor progression and angiogenesis in neuroblastoma in experimental models in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumor growth on active angiogenesis. Here, we present an overview of recent advances in antiangiogenesis in neuroblastoma and describe the most important active substances, preclinical and clinical data, as well as future perspectives.

The poor selectivity of chemotherapeutic drugs for neoplastic cells may lead to dose-limiting side effects that compromise clinical outcomes. Moreover, heterogeneous tumor perfusion and vascular permeability, and increased interstitial pressure, could represent critical barriers that limit the penetration of drugs into neoplastic cells distant from tumor vessels and, consequently, the effectiveness of chemotherapy. We have recently developed two strategies for increasing the local concentration of chemotherapeutic drugs in tumors and their therapeutic index, based on tumor vascular targeting. First, we have found that vascular targeting with minute amounts of tumor necrosis factor alpha (TNF-alpha), an inflammatory cytokine able to increase vascular permeability, alters tumor barriers and increases the penetration of chemotherapeutic drugs in subcutaneous tumors in mouse models. Targeted delivery of TNF-alpha to tumor vessels was achieved by coupling this cytokine with cyclic CNGRC peptide, an aminopeptidase N (CD13) ligand that targets the tumor neovasculature. Second, we have observed that encapsulation of doxorubicin into liposomes able to home to tumor vessels markedly improves drug uptake by neuroblastoma tumors, in an orthotopic xenograft model, and its therapeutic index. Targeted delivery of liposomes was achieved by coupling linear GNGRG peptide to the surface of liposomal doxorubicin. Vascular targeting, either indirectly with NGR-TNF-alpha or directly with NGR-targeted liposomes, could be a novel strategy for increasing the therapeutic index of chemotherapeutic drugs.

Background: Expression of the c-myb proto-oncogene in neuroblastoma, the most common extracranial solid tumor of infancy, is linked with cell proliferation and differentiation. Neuroblastoma can be selectively targeted via monoclonal antibodies against the disialoganglioside (GD2) tumor-associated antigen. Liposomes coated with anti-GD2 antibodies (targeted liposomes) and entrapping a c-myb antisense oligonucleotide have antitumor activity. Because antisense oligonucleotides containing CpG motifs can stimulate immune responses, we evaluated the effect of CpG-containing c-myb antisense oligonucleotides encapsulated within targeted liposomes., Methods: Antisense (myb-as) and scrambled (myb-scr) control oligonucleotides with CpG motifs were encapsulated within GD2-targeted and non-targeted liposomes. Two murine (nude and SCID-bg) xenograft models of neuroblastoma were established. Mice (groups of 10) were injected intravenously with various oligonucleotide and liposome formulations, and life span, long-term survival, immune cell activation, and cytokine release were measured over time., Results: Tumor-bearing mice injected with targeted liposome-CpG-myb-as or targeted liposome-CpG-myb-scr lived longer than mice in any other group, although long-term survival (i.e., more than 120 days) was obtained only in mice injected with targeted liposome-CpG-myb-as. Splenocytes isolated from mice injected with targeted liposome-CpG-myb-as contained activated macrophages, B cells, and natural killer (NK) cells, but only activated NK cells were associated with antitumor cytotoxic activity. In vivo immune cell activation was accompanied by the time-dependent increases in plasma levels of the cytokines interleukin 12 (IL-12; maximum level reached by 2 hours) and interferon gamma (IFN-gamma; maximum level reached by 18 hours) and was dependent on the oligonucleotide CpG motif. Ablation of macrophages or NK cells resulted in a loss of in vivo antitumor activity., Conclusion: Immune cell activation, involving the time-dependent activation of macrophages and NK cells, contributes to the antitumor activity of targeted liposome-CpG-myb-as against neuroblastoma and could improve the effectiveness of antitumor targeted liposomes.

The clinical activity of rituximab has been evaluated in a phase 2 study in both untreated and relapsed mucosa-associated lymphoid tissue (MALT) lymphomas. Treatment consisted of 4 standard (375 mg/m2) weekly doses. Thirty-five patients were enrolled, and 34 completed the treatment program. The primary lymphoma location was stomach in 15 patients, and extragastric in 20. Eleven patients had previously been treated with chemotherapy. At study entry 12 patients had Ann Arbor stage IE, 3 had stage IIE, and 20 had stage IV disease. The overall response rate was 73% (95% confidence interval, 56%-87%), with 15 complete responses and 10 partial responses, and the response rate was significantly higher in the chemotherapy-naive patients, who had an 87% response rate compared with 45% of the previously treated patients (P =.03). The median response duration was 10.5 months. At a median follow-up of 15 months, 9 patients (26%) relapsed. The median time to treatment failure was 14.2 months in the whole series, but it was significantly longer (22 versus 12 months) in the chemotherapy-naive patients compared with those who had prior chemotherapy (P =.001). Most adverse events were of mild to moderate severity with no grade 4 toxicity. This study indicates that rituximab is safe with significant activity in MALT lymphomas.

Purpose: The suitability of neuroblastoma patients for therapy using radiolabeled meta-iodobenzylguanidine (MIBG) is determined by scintigraphy after the administration of a tracer dose of radioiodinated MIBG whose uptake is dependent upon the cellular expression of the noradrenaline transporter (NAT). As a possible alternative to gamma camera imaging, we developed a novel molecular assay of NAT expression. mRNA extracted from neuroblastoma biopsy samples, obtained retrospectively, was reverse transcribed, and NAT-specific cDNA was quantified by real-time PCR, referenced against the expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. EXPERMENTAL DESIGN: Tumor specimens from 54 neuroblastoma patients were analyzed using real-time PCR, and NAT expression was compared with the corresponding diagnostic scintigrams., Results: Forty-eight of 54 (89%) of tumors showed MIBG uptake by scintigraphy. NAT expression was found to be significantly associated with MIBG uptake (P < 0.0001, Fisher's exact test). None of the samples from the six tumors that failed to concentrate MIBG expressed detectable levels of the NAT (specificity = 1.0). However, of the 48 MIBG uptake-positive tumors, only 43 (90%) expressed NAT (sensitivity = 0.9). The real-time PCR test has a positive predictive value of 1.0 but a negative predictive value of 0.55., Conclusions: The results indicate that whereas this method has substantial ability to predict the capacity of neuroblastoma tumors to accumulate MIBG, confirmation is required in prospective studies to determine more accurately the predictive strength of the test and its role in the management of patients with neuroblastoma.

Angiogenesis is a critical event in the progression of human neuroblastoma. This mini-review summarizes our literature and experimental data concerning the use of anti-angiogenic molecules, such as TNP-470 and fenretinide, in neuroblastoma treatment.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. In advanced disease stages, prognosis is poor and treatments have limited efficacy, thus novel strategies are warranted. The synthetic retinoid fenretinide (HPR) induces apoptosis in NB and melanoma cell lines. We reported an in vitro potentiation of HPR effects on melanoma cells when the drug is incorporated into GD2-targeted immunoliposomes (anti-GD2-SIL-HPR). Here, we investigated the antitumor activity of anti-GD2-SIL-HPR against NB cells, both in vitro and in vivo. Anti-GD2-immunoliposomes (anti-GD2-SIL) showed specific, competitive binding to, and uptake by, various NB cell lines. Moreover, anti-GD2-SIL-HPR presented increased selectivity and efficacy in inhibiting NB cell proliferation through the induction of apoptosis, compared to free drug and SL-HPR. In an in vivo NB metastatic model, we demonstrated that anti-GD2-SIL-HPR completely inhibited the development of macroscopic and microscopic metastases in comparison to controls. However, similar, but significantly less potent antitumor effect was observed also in mice treated with anti-GD2 immunoliposomes without HPR (anti-GD2-SIL-blank) or anti-GD2 mAb alone (P=0.0297 and P=0.0294, respectively, vs. anti-GD2-SIL-HPR). Moreover, our results clearly demonstrated that, although anti-GD2 mAb had a strong antitumor effect in this in vivo NB model, 100% curability was obtained only following treatment with anti-GD2-SIL-HPR (P<0.0001). Anti-GD2 liposomal HPR should receive clinical evaluation as adjuvant therapy of neuroblastoma.

The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving signaling pathways mediated by free radicals or reactive oxygen species (ROS). The aim of this study was to identify mechanisms generating ROS and apoptosis of neuroblastoma cells in response to fenretinide. Fenretinide-induced ROS or apoptosis of SH-SY5Y or HTLA 230 neuroblastoma cells were not blocked by Nitro l-argenine methyl ester (l-NAME), an inhibitor of nitric oxide synthase. Flavoprotein-dependent superoxide-producing enzymes such as NADPH oxidase were also not involved in fenretinide-induced apoptosis or ROS generation. Similarly, ketoconazole, a cytochrome P450 inhibitor, and inhibitors of cyclooxygenase (COX) were also ineffective. In contrast, inhibition of phospholipase A(2) or lipoxygenases (LOX) blocked the induction of ROS and apoptosis in response to fenretinide. Using specific inhibitors of LOX, blocking 12-LOX but not 5- or 15-LOX inhibited both fenretinide-induced ROS and apoptosis. The effects of eicosatriynoic acid, a specific 12-LOX inhibitor, were reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid and 12 (S)-hydroxyeicosatetraenoic acid. The targeting of 12-LOX in neuroblastoma cells may thus be a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity., (Copyright 2003 Wiley-Liss, Inc.)

Purpose: Fenretinide (4HPR), a synthetic retinoid, induces apoptosis in neuroblastoma cells. A Phase I study in children with neuroblastoma was designed to determine maximum tolerated dose, toxicity, and pharmacokinetics., Experimental Design: Fifty-four patients received oral 4HPR, once daily, for 28 days, followed by a 7-day interruption, for up to 6 courses. The starting dose was 100 mg/m(2)/day. At least 3 patients were entered at each escalating 4HPR dose level. Pharmacokinetic sampling was performed on days 1 and 28 of the first course., Results: Fifty-four patients, of whom 53 were evaluable, received doses between 100 and 4000 mg/m(2)/day for a total of 168 courses. Additional dose escalation was precluded by capsule number intake. A total of 34 of 53 evaluable patients showed manageable, reversible toxicities, which were not dose related. One dose-limiting toxicity (nyctalopia grade 3) occurred after the 1000 mg/m(2)/day dose. Twelve patients showed grade 2 toxicity: skin xerosis (6 cases); nyctalopia (3 cases); hepatic toxicity (1 case); diarrhea (1 case); and headache (1 case). Stable disease was observed in 41 patients for a median period of 23 months (range 2-35+). After first administration, average 4HPR peak plasma levels ranged from 0.6 to 6 micro M (after 100 and 4000 mg/m(2)/day, respectively) and increased 2-fold (to 1.3 and 12.9 micro M, respectively) after the 28-day treatment. 4HPR half-life increased from 17 h after the first administration to 25 h after the 28(th) administration. Incidence of grade 2-3 toxicity was 0 of 12 (0%), 7 of 22 (31%), and 4 of 8 (50%) with peak 4HPR concentrations <3 micro M, 3-10 micro M, and >10 micro M, respectively. After repeated treatment, retinol levels decreased from 20 to 10% of pretreatment levels after all of the doses., Conclusions: In children, 4HPR administration up to 4000 mg/m(2)/day over 28 days, followed by a 7-day interruption, results in manageable toxicity and in drug plasma concentrations comparable with those that induce apoptosis in neuroblastoma cell lines.

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor- (VEGF-) and fibroblast growth factor-2- (FGF-2)-induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor-induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy-induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF- and FGF-2-specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT-PCR analysis of VEGF and FGF-2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48-72 hr of treatment with HPR, followed by a progressive accumulation of cells in G(1) at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF-2 receptor-2 was reduced after implantation of HPR-loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors., (Copyright 2001 Wiley-Liss, Inc.)

Background: Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro., Methods: Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside GD(2) were covalently coupled to the CCLs. (3)H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis., Results: Our methods produced GD(2)-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to GD(2)-positive neuroblastoma cells that could be blocked by soluble anti-GD(2) monoclonal antibodies. GD(2)-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of fourfold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. GD(2)-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in GD(2)-negative cells., Conclusions: Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells.

Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome-entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10-fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long-circulating GD2-targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti-GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab-bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non-specific isotype-matched Abs or Ab-free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non-specific adsorption of liposomes to the cells, while allowing for maximum Ab-mediated binding. When melanoma cells were incubated with 30 microM HPR entrapped in anti-GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab-free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR.

Melanoma is a highly malignant and increasingly common tumour. Since metastatic melanoma remains incurable, new treatment approaches are needed. Previously, we reported that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide, HPR) induces apoptosis in neuroblastoma cells, sharing a neuroectodermal origin with melanoma cells. Since no data exist thus far on the effects of HPR on human melanoma tumours, our purpose was to investigate the in vitro modulation of cell growth and apoptosis by HPR in melanoma cells. Ten human melanoma cell lines were exposed in vitro to increasing concentrations of HPR. Dose-dependent growth inhibition and cytotoxicity were observed. According to cytofluorimetric analysis, propidium iodide staining and TUNEL assay, HPR-treated melanoma cells were shown to undergo apoptosis. However, IC50 values ranged from 5 to 28 microM, while IC90 values were between 10 and 45 microM. These last concentrations are approximately 10-fold higher than those achievable in patients given oral HPR. To explore the potential of new delivery strategies, HPR was loaded at high concentrations into immunoliposomes directed to disialoganglioside GD2, a tumour-specific antigen extensively expressed by neuroectoderma-derived tumours. Treatment of melanoma cells for a short time (2 hr) with HPR-containing immunoliposomes followed by culture in drug-free medium gave rise to apoptosis of target cells, whereas cells treated for 2 hr with equivalent concentrations of the free drug survived. The efficacy of immunoliposomal HPR was strongly dependent on the density of GD2 expression in the different cell lines.

Iodine-labelled metaiodobenzylguanidine (MIBG) is a radiopharmaceutical used for diagnostic imaging and targeted radiotherapy of neuroendocrine tumors. We previously reported that the ability of a neuroblastoma (NB) cell line, LAN-5, to accumulate MIBG was powerfully stimulated by interferon-gamma (IFN-gamma), a well-known NB differentiation-promoting agent. To extend the above findings, we have investigated 5 NB cell lines for their ability to accumulate 125I-MIBG in basal conditions or after various combinations of differentiative stimuli. Our results show that association of IFN-gamma and tumor necrosis factor-alpha boosts MIBG uptake in the early times of incubation in LAN-5 and GI-LI-N cells, while both SK-N-SH and SK-N-BE(2)c cells are strongly stimulated by co-treatment with IFN-gamma and all-trans retinoic acid. Moreover, although only LAN-5 and GI-LI-N cells are sensitive to IFN-gamma alone, the combination of IFN-gamma and IFN-alpha causes a synergistic increase in MIBG uptake in all the NB cell lines tested. From experiments on MIBG release we conclude that no intracellular storage within specialized structures took place during differentiation. The observed enhancement in MIBG accumulation results from an increased uptake of the drug only. This conclusion was confirmed by analyzing MIBG-transporter gene expression, which was increased in cells subjected differentiative regimens. According to these findings, inducing differentiation of NB cells in vitro appears to improve their MIBG incorporation ability powerfully.

Four human neuroblastoma (NB) cell lines (LAN-5, SK-N-BE(2)C, GI-LI-N, and GI-CA-N) have been investigated for their ability to take up and store [125I]metaiodobenzylguanidine (125I-MIBG) in vitro. Only SK-N-BE(2)C and LAN-5 cells were able to specifically take up MIBG, with the former cell line showing a more efficient retention of the radiotracer. 125I-MIBG incorporation in both cell lines was inhibited by norepinephrine, desipramine, ouabain and energy depletion. Thus, all the major criteria for specific (type 1) uptake were fulfilled. Conversely, GI-LI-N and GI-CA-N cells did not show any specific uptake. Pharmacological manipulations aimed at defining the intracellular site(s) of 125I-MIBG storage clearly showed that the radiotracer is not accumulated in the reserpine-sensitive neurosecretory granules and vesicles in NB cells, contrary to what has been observed in a chromaffin derived tumor cell line (PC12). Our study provides new and suitable models to investigate in vitro the molecular and cellular pharmacology of MIBG in NB cells.

The modalities of uptake and storage of iodine-labeled m-iodobenzylguanidine (MIBG) by four human neuroblastoma cell lines have been studied. SK-N-BE(2)C cell line has been shown to possess the specific (type 1) MIBG uptake, as well as an efficient extravesicular storage mechanism. Conversely, LAN-5 cells, which show a nonsaturation kinetic of MIBG incorporation, lack only the ability to efficiently store the MIBG taken up by a mechanism that can be pharmacologically defined as uptake 1. The two other neuroblastoma cell lines tested, GI-LI-N and GI-CA-N, lack both uptake and storage capacity. In view of the fact that the only detailed study on specific MIBG uptake by a human neuroblastoma cell line has been carried out on SK-N-SH, a highly heterogeneous cell line, our report provides new insights on the molecular and cellular pharmacology of radiolabeled MIBG.

A prospective multicenter program was performed to evaluate the combination of rituximab and high-dose (hd) sequential chemotherapy delivered with multiple autologous peripheral blood progenitor cell (PBPC) support (R-HDS-maps regimen) in previously untreated patients with diffuse large B-cell lymphoma (DLB-CL) and age-adjusted International Prognostic Score (aaIPI) score 2-3. R-HDS-maps includes: (i) three APO courses; (ii) sequential administration of hd-cyclophosphamide (CY), hd-Ara-C, both supplemented with rituximab, hd-etoposide/cisplatin, PBPC harvests, following hd-CY and hd-Ara-C; (iii) hd-mitoxantrone (hd-Mito)/L-Pam + 2 further rituximab doses; (iv) involved-field radiotherapy. PBPC rescue was scheduled following Ara-C, etoposide/cisplatin and Mito/L-Pam. Between 1999 and 2004, 112 consecutive patients aged <65 years (74 score 2, 38 score 3) entered the study protocol. There were five early and two late toxic deaths. Overall 90 patients (80%) reached clinical remission (CR); at a median 48 months follow-up, 87 (78%) patients are alive, 82 (73%) in continuous CR, with 4 year overall survival (OS) and event-free survival (EFS) projections of 76% (CI 68–85%) and 73% (CI 64–81%), respectively. There were no significant differences in OS and EFS between subgroups with Germinal-Center and Activated B-cell phenotype. Thus, life expectancy of younger patients with aaIPI 2-3 DLB-CL is improved with the early administration of rituximab-supplemented intensive chemotherapy compared with the poor outcome following conventional chemotherapy.Leukemia (2007) 21, 1802–1811; doi:10.1038/sj.leu.2404781; published online 7 June 2007 [ABSTRACT FROM AUTHOR]