An unusual presentation of dermatofibrosarcoma protuberans with pleomorphic sarcomatous transformation: potential pitfall and diagnostic strategy.

Dermatofibrosarcoma protuberans (DFSP) is a low grade, superficial sarcoma characterized by a proliferation of monomorphous, spindle cells arranged in a storiform pattern and infiltrating the subcutaneous tissue. The tumor is typically CD34 positive, and shows the characteristic COL1A1-PDGFB fusion gene, detectable either by florescent in situ hybridization (FISH) and polymerase chain reaction (PCR). We describe a case of DFSP with a focus of peculiar pleomorphic sarcomatous transformation. The focus constituted the entire bioptic tissue that was initially excised, raising considerable diagnostic problems for pathologist. The use of FISH as an ancillary technique allowed the right diagnosis.

Atypical Spitz tumors in patients younger than 18 years.

BACKGROUND: Diagnosis and proper management of atypical Spitz tumors in pediatric age are still controversial. OBJECTIVE: We sought to investigate the clinicopathological and molecular features of atypical Spitz tumors in patients aged 18 years or younger. METHODS: We performed a retrospective clinicopathological and fluorescence in situ hybridization study on 50 pediatric atypical Spitz tumors. RESULTS: Parameters that were significantly correlated with a diagnosis of atypical Spitz tumors over Spitz nevus included asymmetry, level IV/V, lack of maturation, solid growth, nuclear pleomorphism, high nuclear-cytoplasmic ratio, atypical and deep mitoses, and more than 6 mitoses/mm(2). In the atypical Spitz tumors group, a significantly higher mitotic rate was observed in prepuberal age (P = .04). The 4-probe fluorescence in situ hybridization melanoma assay did not discriminate atypical Spitz tumors from Spitz nevi. Heterozygous 9p21 loss was found in 3 of 37 cases and homozygous 9p21 loss in 2 of 37 cases. Only 1 child experienced a fatal outcome, showing genetic abnormalities by melanoma fluorescence in situ hybridization probe and a heterozygous 9p21 deletion. LIMITATIONS: The limited number of adverse outcomes did not allow the prognostic analysis of single morphologic features. CONCLUSION: Pediatric atypical Spitz tumors are associated with minimal lethal potential. Atypical Spitz tumors require complete excision and careful follow-up while our data do not support any clinical benefit for the sentinel lymph node biopsy procedure and completion lymphadenectomy.

Logic Learning Machine creates explicit and stable rules stratifying neuroblastoma patients.

BACKGROUND: Neuroblastoma is the most common pediatric solid tumor. About fifty percent of high risk patients die despite treatment making the exploration of new and more effective strategies for improving stratification mandatory. Hypoxia is a condition of low oxygen tension occurring in poorly vascularized areas of the tumor associated with poor prognosis. We had previously defined a robust gene expression signature measuring the hypoxic component of neuroblastoma tumors (NB-hypo) which is a molecular risk factor. We wanted to develop a prognostic classifier of neuroblastoma patients' outcome blending existing knowledge on clinical and molecular risk factors with the prognostic NB-hypo signature. Furthermore, we were interested in classifiers outputting explicit rules that could be easily translated into the clinical setting. RESULTS: Shadow Clustering (SC) technique, which leads to final models called Logic Learning Machine (LLM), exhibits a good accuracy and promises to fulfill the aims of the work. We utilized this algorithm to classify NB-patients on the bases of the following risk factors: Age at diagnosis, INSS stage, MYCN amplification and NB-hypo. The algorithm generated explicit classification rules in good agreement with existing clinical knowledge. Through an iterative procedure we identified and removed from the dataset those examples which caused instability in the rules. This workflow generated a stable classifier very accurate in predicting good and poor outcome patients. The good performance of the classifier was validated in an independent dataset. NB-hypo was an important component of the rules with a strength similar to that of tumor staging. CONCLUSIONS: The novelty of our work is to identify stability, explicit rules and blending of molecular and clinical risk factors as the key features to generate classification rules for NB patients to be conveyed to the clinic and to be used to design new therapies. We derived, through LLM, a set of four stable rules identifying a new class of poor outcome patients that could benefit from new therapies potentially targeting tumor hypoxia or its consequences.

Peripheral neuroblastic tumors with genotype-phenotype discordance: a report from the Children's Oncology Group and the International Neuroblastoma Pathology Committee.

BACKGROUND: Of 4,706 peripheral neuroblastic tumors (pNTs) registered on the Children's Cancer Group and Children's Oncology Group Neuroblastoma Study between 1989 and 2010, 51 cases (1.1%) had genotype-phenotype discordance characterized by MYCN amplification (indicating poor prognosis) and Favorable Histology (indicating better prognosis). PROCEDURE: To distinguish prognostic subgroups in the genotype-phenotype discordant pNTs, two subgroups, "conventional" and "bull's eye," were identified based on the nuclear morphology. The "conventional" tumors (35 cases) included: Neuroblastoma, poorly differentiated subtype (NB-PD, 26 cases) with "salt-and-pepper" nuclei; neuroblastoma, differentiating subtype (4 cases); ganglioneuroblastoma, intermixed (3 cases); and ganglioneuroma, maturing subtype (2 cases). The "bull's eye" tumors included NB-PD with prominent nucleoli (16 cases). Clinicopathologic characteristics of these two subgroups were analyzed. N-myc protein expression was tested immunohistochemically on available tumors. RESULTS: No significant difference was found between these two subgroups in the distribution of prognostic factors such as age at diagnosis, clinical stage, histopathology category/subtype, mitosis-karyorrhexis index, ploidy, 1p LOH, and unbalanced 11q LOH. However, prognosis of the patients with "conventional" tumors (5-year EFS 85.7 ± 12.2%; OS 89.3 ± 10.3%) was significantly better than those with "bull's eye" tumors (EFS 31.3 ± 13.0%; OS 42.9 ± 16.2%; P = 0.0010 and 0.0008, respectively). Immunohistochemically all (11/11) tested "conventional" tumors were negative, and 10/11 tested "bull's eye" tumors were positive for N-myc protein expression. CONCLUSIONS: Based on the presence or absence of prominent nucleoli (the putative site of RNA synthesis/accumulation leading to N-myc protein expression), two prognostic subgroups, "conventional" with a better prognosis and "bull's eye" with a poor prognosis, were distinguished among the genotype-phenotype discordant pNTs.

Interferon-γ-dependent inhibition of B cell activation by bone marrow-derived mesenchymal stem cells in a murine model of systemic lupus erythematosus.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered a promising tool for the treatment of immune-mediated diseases. This study was undertaken to assess the influence of murine BM-MSCs on the activation of B cells in (NZB × NZW)F(1) mice as an animal model of systemic lupus erythematosus (SLE). METHODS: We evaluated the in vitro effects of BM-MSCs on the proliferation and differentiation to plasma cells of splenic mature B cell subsets, namely follicular and marginal zone B cells isolated from (NZB × NZW)F(1) mice. Lupus mice were also treated with BM-MSCs, and serum autoantibodies, proteinuria, histologic changes in the kidney, and survival rates were monitored. RESULTS: BM-MSCs inhibited antigen-dependent proliferation and differentiation to plasma cells of follicular and marginal zone B cells in vitro. This inhibitory effect was dependent on interferon-γ (IFNγ) and was mediated by cell-to-cell contact, involving the programmed death 1 (PD-1)/PD ligand pathway. In vivo treatment with BM-MSCs did not affect the levels of anti-double-stranded DNA antibodies or proteinuria. However, a reduction in glomerular immune complex deposition, lymphocytic infiltration, and glomerular proliferation was observed. CONCLUSION: Our findings indicate that BM-MSCs affect B cell receptor-dependent activation of both follicular and marginal zone B cells from lupus mice. This inhibitory effect is IFNγ-dependent and cell contact-dependent. MSCs in vivo do not affect the production of autoantibodies, the level of proteinuria, or the mortality rates. Nonetheless, the significant improvement in histologic findings in the kidney supports the potential role of MSCs in the prevention of glomerular damage.

Identification of multiple hypoxia signatures in neuroblastoma cell lines by l1-l2 regularization and data reduction.

Hypoxia is a condition of low oxygen tension occurring in the tumor and negatively correlated with the progression of the disease. We studied the gene expression profiles of nine neuroblastoma cell lines grown under hypoxic conditions to define gene signatures that characterize hypoxic neuroblastoma. The l(1)-l(2) regularization applied to the entire transcriptome identified a single signature of 11 probesets discriminating the hypoxic state. We demonstrate that new hypoxia signatures, with similar discriminatory power, can be generated by a prior knowledge-based filtering in which a much smaller number of probesets, characterizing hypoxia-related biochemical pathways, are analyzed. l(1)-l(2) regularization identified novel and robust hypoxia signatures within apoptosis, glycolysis, and oxidative phosphorylation Gene Ontology classes. We conclude that the filtering approach overcomes the noisy nature of the microarray data and allows generating robust signatures suitable for biomarker discovery and patients risk assessment in a fraction of computer time.

Medulloblastoma variants: age-dependent occurrence and relation to Gorlin syndrome--a new clinical perspective.

PURPOSE: We aimed to test the hypothesis that medulloblastoma (MB) variants show a different age distribution and clinical behavior reflecting their specific biology, and that MB occurring at very young age is associated with cancer predisposition syndromes such as Gorlin syndrome (GS). EXPERIMENTAL DESIGN: We investigated the frequency, age distribution, location, response to treatment, outcome, and association with familial cancer predisposition syndromes in a series of 82 cases of MB in patients ages <14 years diagnosed at the Giannina Gaslini Children's Hospital, Genoa, between 1987 and 2004. RESULTS: Desmoplastic MB and MB with extensive nodularity (MBEN), were present in 22 of 82 cases (27%) and were more frequent in children ages

The tumor suppressor gene TRC8/RNF139 is disrupted by a constitutional balanced translocation t(8;22)(q24.13;q11.21) in a young girl with dysgerminoma.

BACKGROUND: RNF139/TRC8 is a potential tumor suppressor gene with similarity to PTCH, a tumor suppressor implicated in basal cell carcinomas and glioblastomas. TRC8 has the potential to act in a novel regulatory relationship linking the cholesterol/lipid biosynthetic pathway with cellular growth control and has been identified in families with hereditary renal (RCC) and thyroid cancers. Haploinsufficiency of TRC8 may facilitate development of clear cell-RCC in association with VHL mutations, and may increase risk for other tumor types. We report a paternally inherited balanced translocation t(8;22) in a proposita with dysgerminoma. METHODS: The translocation was characterized by FISH and the breakpoints cloned, sequenced, and compared. DNA isolated from normal and tumor cells was checked for abnormalities by array-CGH. Expression of genes TRC8 and TSN was tested both on dysgerminoma and in the proposita and her father. RESULTS: The breakpoints of the translocation are located within the LCR-B low copy repeat on chromosome 22q11.21, containing the palindromic AT-rich repeat (PATRR) involved in recurrent and non-recurrent translocations, and in an AT-rich sequence inside intron 1 of the TRC8 tumor-suppressor gene at 8q24.13. TRC8 was strongly underexpressed in the dysgerminoma. Translin is underexpressed in the dysgerminoma compared to normal ovary.TRC8 is a target of Translin (TSN), a posttranscriptional regulator of genes transcribed by the transcription factor CREM-tau in postmeiotic male germ cells. CONCLUSION: A role for TRC8 in dysgerminoma may relate to its interaction with Translin. We propose a model in which one copy of TRC8 is disrupted by a palindrome-mediated translocation followed by complete loss of expression through suppression, possibly mediated by miRNA.

Presence of 1q gain and absence of 7p gain are new predictors of local or metastatic relapse in localized resectable neuroblastoma.

We have addressed the search of novel genetic prognostic markers in a selected cohort of patients with stroma-poor localized resectable neuroblastoma (NB) who underwent relapse or progression (group 1) or complete remission (group 2) over a minimum follow-up of 32 months from diagnosis. Twenty-three Italian patients with localized resectable NB (stages 1 and 2) diagnosed from 1994 through 2005 were studied. All patients received surgical treatment. Chemotherapy was administered only to the three stage 2 patients who had MYCN-amplified tumors. High-resolution array-comparative genomic hybridization (CGH) DNA copy-number analysis technology was used to identify novel prognostic markers. Chromosome 1p36.22p36.32 loss and 1q22qter gain, detected almost exclusively in group 1 patients, were significantly associated with poor event-free survival (EFS) (p = 0.0024 and p = 0.024, respectively). In contrast, patients with 7p11.2p22 gain, who belonged predominantly to group 2, had a significantly better EFS (p = 0.015). The frequency of 17q gain or 3p and 11q losses did not differ significantly in group 1 versus group 2 NBs. The sensitive technique allowed us to define the smallest region of 1p deletion. In conclusion, 1q22qter gain and 7p11.2p22 gain might represent new prognostic markers in localized resectable NB, but the small study size and the retrospective nature of the findings warrant further validation of the results in larger studies.

Multiple relapses of visceral leishmaniasis in an adolescent with idiopathic CD4+ lymphocytopenia associated with novel immunophenotypic and molecular features.

An adolescent with idiopathic CD4 lymphocytopenia suffered from 4 visceral leishmaniasis relapses despite appropriate treatment. CD8 lymphocytopenia and abnormal expansion of TCRalphabeta, CD4, CD8 cells were consistently detected together with reduced export of mature T cells from thymus. This novel form of idiopathic CD4 lymphocytopenia may predispose to multiple visceral leishmaniasis relapses.

Expression and functional analysis of human leukocyte antigen class I antigen-processing machinery in medulloblastoma.

Defects in the expression and/or function of the human leukocyte antigen (HLA) class I antigen-processing machinery (APM) components are found in many tumor types. These abnormalities may have a negative impact on the interactions of tumor cells with host's immune system and on the outcome of T cell-based immunotherapy. To the best of our knowledge, no information is available about APM component expression and functional characteristics in human medulloblastoma cells (Mb). Therefore, in the present study, we have initially compared the expression of APM components in Mb, an embryonal pediatric brain tumor with a poor prognosis, with that in noninfiltrating astrocytic pediatric tumors, a group of differentiated brain malignancies with favorable prognosis. LMP2, LMP7, calnexin, beta2-microglobulin-free heavy chain (HC) and beta2-microglobulin were down-regulated or undetectable in Mb lesions, but not in astrocytic tumors or normal fetal cerebellum. Two Mb cell lines (DAOI and D283) displayed similar but not superimposable defects in APM component expression as compared with primary tumors. To assess the functional implications of HLA class I APM component down-regulation in Mb cell lines, we tested their recognition by HLA class I antigen-restricted, tumor antigen (TA)-specific CTL, generated by stimulations with dendritic cells that had been transfected with Mb mRNA. The Mb cell lines were lysed by TA-specific CTL in a HLA-restricted manner. Thus, defective expression of HLA class I-related APM components in Mb cells does not impair their ability to present TA to TA-specific CTL. In conclusion, these results can contribute to optimize T cell-based immunotherapeutic strategies for Mb treatment.

Identification of novel chromosomal abnormalities and prognostic cytogenetics markers in intracranial pediatric ependymoma.

Aim of this study was to search for novel chromosomal imbalances and potential prognostic markers in pediatric ependymoma. Tumor DNA, obtained from 20 children with intracranial ependymoma (World Health Organization WHO grades II and III), was analyzed using metaphase-based comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH). The novel copy number aberrations (CNAs) here identified are (i) 4q33-qter loss, (ii) 10q25.2-q26.3 gain, (iii) 3q23-qter losses, (iv) 18q22.2 loss, and (v) 19p13.1-p13.3 gain. The combined presence of 6p22-pter and 13q14.3-qter losses predicted significantly reduced survival. Larger studies are warranted to validate these findings.

Eosinophilic chromophobe renal cell carcinoma in a child with hypospadias.

Chromophobe renal cell carcinoma (CRCC) is a distinct variant of renal carcinoma generally affecting adults. We report a case of an unusual CRCC, arising in a male child affected by hypospadias. This case demonstrates that CRCC can occur in the pediatric patients and can be associated with genital tract anomalies such as Wilms tumor.

Diagnostic value of serological assays in pediatric inflammatory bowel disorders.

BACKGROUND: Pediatric studies reported that the combined use of the anti-neutrophil cytoplasm autoantibodies (ANCA) and the anti-Saccharomyces cerevisiae mannan antibodies (ASCA) may be a specific useful noninvasive test in the diagnosis of inflammatory bowel diseases (IBD). AIMS: To evaluate the diagnostic accuracy of ANCA and ASCA in children with suspected IBD, and to see whether different commercially available assays (indirect immunofluorescence vs. ELISA) agree well enough in terms of analytical performance. PATIENTS AND METHODS: Sixty-nine children (30 males, 39 females, age range 2-18 years) with suspicion of IBD entered the study. Before colonoscopy, a blood sample was also drawn to assess ASCA and ANCA. RESULTS: A diagnosis of IBD was established in 47 patients; the remainder had infective or other causes of colitis. For ulcerative colitis, the association ASCA-/ANCA+ had 70% sensitivity and 86% specificity, with a positive predictive value of 82%. The association ASCA+/ANCA- had 86% sensitivity and 93% specificity for Crohn's disease, with a positive predictive value of 75%. CONCLUSION: Although more experience is needed to state the diagnostic power of serologic assay, determination of ANCA and ASCA in IBD children may help both in distinguish these conditions from other entities and ulcerative colitis from Crohn's disease, particularly in doubtful cases.

Rhinopharyngeal B-cell non-hodgkin lymphoma: unusual presentation with massive hematemesis.

Lymphomas are the third most frequent type of childhood cancer after acute leukemias and brain tumors. Symptoms at diagnosis are extremely different depending on several factors such as histological subtype, disease extent, and site of tumor. We report an unusual presentation describing a case of epistaxis of the posterior nasal fossa with severe hematemesis.

Multiple defects of the antigen-processing machinery components in human neuroblastoma: immunotherapeutic implications.

Low expression of human leukocyte antigen (HLA) class I in human tumors may be related to defects of the antigen-processing machinery (APM) components. Neuroblastoma cells are virtually HLA class I negative, but (i) the underlying mechanisms are unknown, and (ii) expression of the APM components has never been investigated. Here we have used a panel of novel monoclonal antibodies to proteasomal and immunoproteasomal components, chaperons and transporter associated with antigen processing (TAP) to characterize 24 stroma-poor neuroblastoma tumors and six neuroblastoma cell lines. Primary tumors showed defects in the expression of zeta, tapasin, TAP1 or TAP2, HLA class I heavy chain and beta2 microglobulin, LMP2 and LMP7, as compared to normal adrenal medulla. Neuroblastoma cell lines displayed roughly similar patterns of APM expression in comparison to primary tumors. Incubation of neuroblastoma cell lines with interferon-gamma caused upregulation of HLA class I molecules and reduced lysis by killer inhibitory receptor HLA ligand-matched NK cells. Defects in APM components explain reduced peptide loading on HLA class I molecules, their instability and failure to be expressed on the cell surface. HLA class I upregulation by interferon-gamma, although enhancing neuroblastoma cell recognition by cytotoxic T cells, dampens their susceptibility to NK cells.

Diagnostic identification of malignant cells in the cerebrospinal fluid by tumor-specific qRT-PCR.

Tumor specific quantitative RT-PCRs for two neuroblastoma specific molecular markers, tyrosine hydroxylase (TH) and GD2 synthase, were used to unequivocally demonstrate the neoplastic nature of the cells present in the cerebrospinal fluid of a neuroblastoma patient. After radical surgery of two separate tumoral lesions, localized in the extradural area, the patient presented with meningitis. Common sites of neuroblastoma metastatization, e.g. bone and bone marrow, were not infiltrated by tumor cells, as assessed by standard scintigraphy, morphological investigation and by sensitive and specific immunocytochemical and molecular assays. The results presented here demonstrate the successful use of tumor-specific qRT-PCRs in cerebrospinal fluid to investigate questionable clinical cases. The technique, which compared to other detection methods (e.g., immunocytochemistry) requires very few cells, yields unambiguous information once a suspected diagnosis has been formulated and a tumor-specific molecular marker is available.

Chromosomal imbalances in pediatric Burkitt-like lymphoma and review of the literature in relation to other germinal center derived B-cell tumors.

This study reports on the cytogenetic features of a novel case of pediatric Burkitt-like lymphoma (BLL), that adds to the three published. Four groups of cytogenetic abnormalities were detected in the present case: (1) imbalances shared by most germinal center (GC) derived B-cell tumors including BLL (+1q, -6q, -8p, +8q24 and +11); (2) imbalances already reported in adult but not in pediatric BLL cases and shared with most GC B-cell tumors (+7, -9p, -9q, +12q, -13q, +17, +19, -3 and -4); (3) imbalances already reported in pediatric but not in adult BLL cases and shared with some GC B-cell tumors (-2q); and (4) imbalances never described before in pediatric or adult BLL, but present in different GC B-cell tumors (-6p, -1p and -18q). In view of the paucity of pediatric BLL cases published, this report adds novel, relevant information on the molecular cytogenetic features of this rare tumor.

Sequential immunogene therapy with interleukin-12- and interleukin-15-engineered neuroblastoma cells cures metastatic disease in syngeneic mice.

PURPOSE: To investigate the potential synergistic effects of Neuro2a neuroblastoma cells engineered with IL-12 and/or IL-15 genes in improving survival of syngeneic mice bearing neuroblastoma metastatic disease. EXPERIMENTAL DESIGN: Neuro2a cells engineered with interleukin (IL)-12 (Neuro2a/IL-12), IL-15 (Neuro2a/IL-15), or both cytokines (Neuro2a/IL-12/IL-15) were injected s.c. in syngeneic A/J mice challenged i.v. with Neuro2a parental cells (Neuro2apc) using different schedules of administration in either preventive or therapeutic settings. RESULTS: A single injection of Neuro2a/IL-12 or Neuro2a/IL-15 cells induced resistance to a subsequent i.v. Neuro2apc challenge in 45% and 28% of mice, respectively. Neuro2a/IL-12/IL-15 cells protected 28% of mice, showing no synergistic effect. However, sequential vaccination with Neuro2a/IL-12 (day -30) followed by Neuro2a/IL-15 (day -15) protected 71% of mice from subsequent challenge with Neuro2apc. A single dose of Neuro2a/IL-12 prolonged the mean survival time of mice bearing established metastatic neuroblastoma from 21 +/- 3 to 46 +/- 27 days but failed to cure mice, whereas Neuro2a/IL-15 or Neuro2a/IL-12/IL-15 were ineffective. However, sequential vaccination with Neuro2a/IL-12 (day +3) followed by Neuro2a/IL-15 (day +13) cured 43% of mice as assessed by histologic analysis of different organs from long-term surviving mice. CTL activity against Neuro2apc cells was observed in splenocytes from treated mice, and CD8(+) T-cell depletion abrogated the therapeutic effect of vaccination. CONCLUSIONS: Sequential vaccination with IL-12- and IL-15-engineered neuroblastoma cells induced optimal preventive and therapeutic effects, which may be related to the Th1 priming effect of IL-12 followed by the enhancement of CD8(+) T-cell responses and their maintenance mediated by IL-15.

Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposomal chemotherapy.

The poor selective toxicity of chemotherapeutic anticancer drugs leads to dose-limiting side effects that compromise clinical outcome. Solid tumors recruit new blood vessels to support tumor growth, and unique epitopes expressed on tumor endothelial cells can function as targets for the anti-angiogenic therapy of cancer. An NGR peptide that targets aminopeptidase N, a marker of angiogenic endothelial cells, was coupled to the surface of liposomal doxorubicin (NGR-SL[DXR]) and was used to treat orthotopic neuroblastoma (NB) xenografts in SCID mice. Pharmacokinetic studies indicated that liposomes coupled to NGR peptide had long-circulating profiles in blood. Their uptake into NB tumor was time dependent, being at least 10 times higher than that of nontargeted liposomes (SL[DXR]) after 24 h, with DXR spreading outside the blood vessels and into the tumors. No uptake was observed into tumors of mice treated with the mismatched peptide ARA-targeted SL[DXR]. Tumor-specific DXR uptake was completely blocked when mice were coinjected with a 50-fold molar excess of the soluble NGR peptide. Adrenal tumor-bearing mice treated with 2 mg/kg/week/x3 of NGR-SL[DXR] partly outlived the control mice (P < 0.001), whereas doses > 3 mg/kg/week/x3 were toxic. Histopathological analysis of cryosections taken from treated mice revealed pronounced destruction of the tumor vasculature with a marked decreased in vessel density. Double staining of tumors with terminal deoxynucleotidyl transferase-mediated nick end labeling and antifactor VIII antibody or antihuman NB demonstrated endothelial cell apoptosis in the vasculature, as well as increased tumor cell apoptosis. Moreover, mice injected with 3 mg/kg/week/x3 of NGR-SL[DXR] displayed rapid tumor regression, as well as inhibition of metastases growth (P = 0.0002). One day after the third treatment, four of six mice showed no evidence of tumors, and the two others showed a >80% reduction in tumor mass and a >90% suppression of blood vessel density (P < 0.01). In contrast, mice treated with ARA-SL[DXR] formed large well-vascularized tumors. Finally, a metronomic administration of NGR-SL[DXR] (1 mg/kg/every other 2 days x 9) induced complete tumor eradication in all animals (P < 0.0001). Our strategy markedly enhanced the therapeutic index of DXR and enabled metronomic administration of therapeutic doses. A dual mechanism of action is proposed: indirect tumor cell kill via the destruction of tumor endothelium by NGR-targeted liposomes and direct tumor cell kill via localization of liposomal DXR to the tumor interstitial space. This combined strategy has the potential to overcome some major limitations of conventional chemotherapy.