Integrated functional and spatial profiling of tumour immune responses induced by immunotherapy: the iPROFILER platform.

BACKGROUND: Cancer immunotherapy elicits functional activation and changes in immune cell distribution in cancer. Tumour heterogeneity is a reason for treatment failure but is difficult to capture in experimental settings. This proof-of-principle study describes the integrated functional and digital spatial profiling platform iPROFILER to capture in-situ immune activation patterns with high precision. MATERIALS AND METHODS: iPROFILER combines an algorithm-based image analysis approach for spatial profiling with functional analyses of patient-derived tumour fragments (PDTFs). This study utilized a folate receptor 1 (FOLR1)xCD3 bispecific antibody in dual-affinity re-targeting (DART) format as a tool for inducing T-cell responses in patient tumour samples, and an in-depth investigation of the immune perturbations induced in the tumour microenvironment was performed. RESULTS: Ex-vivo DART stimulation induces upregulation of multiple activation markers in CD4+ and CD8+ T-cell populations and secretion of pro-inflammatory cytokines in FOLR1-positive tumour specimens. This response was reduced or absent in tissue samples that did not express FOLR1. Immunological responses were driven by a strong induction of interferon gamma (IFNγ) and IFNγ-induced chemokines suggestive of activation of cytotoxic or Th1-like T cells. Ex-vivo DART treatment led to a numerical increase in effector T cells and an upregulation of immune activation markers in the tumour microenvironment as captured by digital image analysis. Analysis of immune activation in tumour and stromal regions further supported the potential of the platform to measure local differences in cell-type-specific activation patterns. CONCLUSIONS: iPROFILER effectively combines functional and spatial readouts to investigate immune responses ex vivo in human tumour samples.

S3-Guideline non-traumatic adult femoral head necrosis.

INTRODUCTION: The treatment of adult non-traumatic avascular necrosis of the femoral head (AVN; N-ANFH) within an estimated incidence of 5000-7000 cases per annum in Germany remains a challenge. Risk factors include steroids, alcohol abuse, chemotherapy and immunosuppressive medication, but a genetic predisposition has been suggested. Early diagnosis of this often bilateral disease process is essential for successful conservative or joint preserving surgical management. In this review, we present the update German consensus S3 guideline "diagnosis and management for N-ANFH" as a concise summary. MATERIALS AND METHODS: This systematic review is based on the published literature from January 1, 1970 to April 31, 2013 (German and English language). Inclusion criteria were systematic reviews, meta-analyses and relevant peer review publications. We identified a total of 3715 related publications, of which 422 were suitable according to the SIGN criteria, but only 159 fulfilled our inclusion criteria. RESULTS AND CONCLUSIONS: Clinical suspicion of N-ANFH mandates radiographic evaluation. If radiographs are normal MRI scans are recommended, which should be evaluated according to the ARCO-classification. Differential diagnoses include transient osteoporosis, bone bruise, insufficiency fracture and destructive arthropathy. Untreated, subchondral fractures commonly occur within 2 years, during which the risk for contralateral involvement is high-thereafter unlikely. Conservative management with Ilomedin and Alendronat can be tried, but other pharmacological or physical treatments are inappropriate. No specific joint preserving procedure can be recommended, but core decompression should be considered in early stages if necrosis is <30 %. In ARCO stages IIIc or IV total hip arthroplasty (THA) should be contemplated, which offers similar outcome compared to osteoarthritis. Young age is the main risk factor for higher revision rates after THA for N-ANFH.

[S3 Guideline. Part 1: Diagnosis and Differential Diagnosis of Non-Traumatic Adult Femoral Head Necrosis]. / S3-Leitline. Teil 1: Diagnostik und Differenzialdiagnostik der atraumatischen Femurkopfnekrose (aFKN) des Erwachsenen.

Non-traumatic femoral head necrosis (FHN) is primarily a disease of the middle-aged adult. Early diagnosis, at a time with lacking or minimal clinical symptoms, is mandatory to consider conservative therapy or joint preserving operations as a therapeutic option. The new German S3 guideline about diagnosis and therapy of FHN is a cooperative effort of five professional medical societies, overall headed by the Deutsche Gesellschaft für Orthopädie und Orthopädische Chirurgie (DGOOC). This review (part I/III) cites and explains the statements of the S3 guideline as agreed on the use of imaging methods for diagnosis of FHN. A diagnostic algorithm is presented. FHN clinically has to be considered in case of equivocal pain of a hip joint with a minimum of 6 weeks duration, when risk factors can be revealed, groin pain at clinical investigation, limping, pain or limitation of movement in case of load, and no obvious differential diagnoses. Is an FHN clinically suspected, primarily radiographs of the pelvis ap and a Lauenstein projection of the hip involved should be carried out. When the radiographs are normal, an MRI of the hips should follow routinely. MRI allows the diagnosis of FNH with high accuracy. Furthermore, MRI reveals the site and the size of the necrotic area involved and evaluates the integrity of the joint surface and subchondral fractures. When ARCO stage II (ARCO: Association Research Circulation Osseous) is diagnosed and MRI does not allow one to determine the joint surface with certainty, a CT of the hip joints should be performed. The S3 guideline explains and recommends the use of the ARCO classification. Although, this classification of 1993 is still largely based on radiographs, the pragmatic use of an "extended" version seems reasonable. Today, classical radiographic criteria like impression of the joint surface and subchondral fractures ("crescent sign") are better to be evaluated by MRI, in cases of subtle findings MRI is even surpassed by CT. The extent of the necrosis in the femoral head as well as the size of the surface area involved is best revealed with MRI. Additionally, in the era of cross sectional imaging a stage "0" seems obsolete. The guideline also addresses practically important considerations about the differential diagnosis of misleading MRI findings. This especially holds true for bone marrow oedema in the femoral head which may be misinterpreted. The differentiating features between FHN, transient bone marrow oedema and destructive arthropathy are discussed.

[S3 Guideline. Part 3: Non-Traumatic Avascular Necrosis in Adults - Surgical Treatment of Atraumatic Avascular Femoral Head Necrosis in Adults]. / S3-Leitlinie. Teil 3: Atraumatische Femurkopfnekrose des Erwachsenen - Operative Therapie der atraumatischen Femurkopfnekrose des Erwachsenen.

The present article describes the guidelines for the surgical treatment of atraumatic avascular necrosis (aFKN). These include joint preserving and joint replacement procedures. As part of the targeted literature, 43 publications were included and evaluated to assess the surgical treatment. According to the GRADE and SIGN criteria level of evidence (LoE), grade of recommendation (EC) and expert consensus (EK) were listed for each statement and question. The analysed studies have shown that up to ARCO stage III, joint-preserving surgery can be performed. A particular joint-preserving surgery currently cannot be recommended as preferred method. The selection of the method depends on the extent of necrosis. Core decompression performed in stage ARCO I (reversible early stage) or stage ARCO II (irreversible early stage) with medial or central necrosis with an area of less than 30 % of the femoral head shows better results than conservative therapy. In ARCO stage III with infraction of the femoral head, the core decompression can be used for a short-term pain relief. For ARCO stage IIIC or stage IV core decompression should not be performed. In these cases, the indication for implantation of a total hip replacement should be checked. Additional therapeutic procedures (e.g., osteotomies) and innovative treatment options (advanced core decompression, autologous bone marrow, bone grafting, etc.) can be discussed in the individual case. In elective hip replacement complications and revision rates have been clearly declining for decades. In the case of an underlying aFKN, however, previous joint-preserving surgery (osteotomies and grafts in particular) can complicate the implantation of a THA significantly. However, the implant life seems to be dependent on the aetiology. Higher revision rates for avascular necrosis are particularly expected in sickle cell disease, Gaucher disease, or kidney transplantation patients. Furthermore, the relatively young age of the patient with avascular necrosis should be seen as the main risk factor for higher revision rate. The results after resurfacing (today with known restricted indications) and cemented as well as cementless THA in aFKN are comparable for the appropriate indication to those in coxarthrosis or other diagnoses. Regardless of the underlying disease endoprosthetic treatment in aFKN leads to good results. Both cemented and cementless fixation techniques can be recommended.

[S3 guideline. Part 2: Non-Traumatic Avascular Femoral Head Necrosis in Adults - Untreated Course and Conservative Treatment]. / S3-Leitlinie. Teil 2: Atraumatische Femurkopfnekrose des Erwachsenen - unbehandelter Verlauf und konservative Behandlung.

BACKGROUND: In Germany there are 5000 to 7000 new cases of atraumatic avascular necrosis of the femoral head in adults per year. It occurs mostly in middle age. An increased frequency of idiopathic cases can be observed. Chemotherapy, corticoids and kidney transplants are frequently associated with the disease. In most cases the disease occurs on both sides. Early diagnosis is of particular importance, since in early stages it is most likely to avoid late damage with joint destruction. Whereas previously the temporary operational joint preservation and subsequent joint replacement were often the only option of treatment, conservative and joint-preserving measures today play an increasing role. MATERIAL AND METHODS: After the AWMF guidelines for S3 guideline clinical questions were formulated. Over the period from 01/01/1970 to 31/05/2013 a literature search was conducted. Systematic reviews, metaanalyses, original papers and clinical trials of all designs were evaluated. There were a total of 3715 references, of which 422 for the assessment regarding SIGN were eligible and finally 180 were in accord with the defined inclusion and exclusion criteria. For the untreated course and the assessment of conservative measures, a total of 42 references was suitable. In formulating the recommendations the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) system was used, which distinguishes A "shall", B "should" and 0 "can". RESULTS AND CONCLUSION: If left untreated, the aFKN within 2 years leads to a subchondral fracture and subsequent collapse. After the diagnosis of femoral head necrosis, the risk of a disease of the opposite side is high within the next 2 years, then unlikely. The sole conservative treatment brings no benefit for the treatment of atraumatic avascular necrosis in the adult. Although it improves function, less pain can be obtained, and surgical intervention can be delayed, the progression is not stopped. Conservative treatment must therefore always be part of the overall treatment. In ARCO stage I to II Iloprost may be considered as a pharmacological approach to reduce the pain and the bone marrow oedema. This also applies to alendronate. Since this is an off-label use, and thus a therapeutic trial, an appropriate patient education must take place. For the use of anticoagulants and statins, there is no recommendation. Also the hyperbaric oxygen therapy, shock waves and pulsating electromagnetic fields or electrical stimulation cannot be recommended.

[Osteomalacia and vitamin D deficiency]. / Osteomalazie und Vitamin-D-Hypovitaminose.

BACKGROUND: Vitamin D and calcium deficiency has a higher incidence in the orthopedic-trauma surgery patient population than generally supposed. In the long term this can result in osteomalacia, a form of altered bone mineralization in adults, in which the cartilaginous, non-calcified osteoid does not mature to hard bone. AIM: The current value of vitamin D and its importance for bones and other body cells are demonstrated. RESULTS: The causes of vitamin D deficiency are insufficient sunlight exposure, a lack of vitamin D3 and calcium, malabsorption, and rare alterations of VDR signaling and phosphate metabolism. The main symptoms are bone pain, fatigue fractures, muscular cramps, muscle pain, and gait disorders, with an increased incidence of falls in the elderly. Osteopathies induced by pharmaceuticals, tumors, rheumatism or osteoporosis have to be considered as the main differential diagnoses. CONCLUSIONS: In addition to the recording of symptoms and medical imaging, the diagnosis of osteomalacia should be ensured by laboratory parameters. Adequate treatment consists of the high-dose intake of vitamin D3 and the replacement of phosphate if deficient. Vitamin D is one of the important hormone-like vitamins and is required in all human cells. Deficiency of vitamin D has far-reaching consequences not only for bone, but also for other organ systems.

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia.

Selective cytotoxicity to cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Here we developed a tumor antigen-targeted delivery of immunonanoparticle carrying a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A (PP2A), phosphorylation and nuclear translocation of SHP1(S591) and deregulation of multiple cellular processes in chronic lymphocytic leukemia (CLL) resulting in potent cytotoxicity. To preclude OSU-2S-mediated effects on these ubiquitous phosphatases in unintended cells and avoid potential adverse effects, we developed an OSU-2S-targeted delivery of immunonanoparticles (2A2-OSU-2S-ILP), that mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 (hROR1) in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL directed immunonanoparticles and the novel transgenic (Tg) mouse model of CLL that expresses hROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia.

[Standards and perspectives for thromboembolism prophylaxis]. / Standards und Perspektiven der Thromboseprophylaxe.

Thromboembolic complications are one of the most severe complications after orthopaedic or trauma surgery. More than 50% of patients undergoing total knee replacement are at risk of suffering deep-vein thrombosis if not provided sufficient prophylaxis. The former standard prophylaxis with unfractionated heparin has been changed over the few last years to low molecular weight heparin or heparinoids, due to the increased incidence of heparin-induced thrombocytopenia under therapy with unfractionated heparin. Risk management is based on different risk levels: highest risk, high risk, intermediate risk and low risk. The probabilities of suffering from deep-vein thrombosis have been determined dependent on the risk level. In patients with total knee replacement, which are at highest risk, a higher dose for the prevention of thromboembolism has been recommended. The synthetic, selective antithrombin-binding pentasaccharide fondaparinux has been successfully used in prophylaxis for the prevention of thrombosis in highest risk patients. However, because of a higher risk of bleeding, this pentasaccharide can be only given 6-8 h after surgery. Low molecular weight heparins and the pentasaccharide are the standard pharmacological prophylaxis for the prevention of venous thromboembolism. Physical therapy, pneumatic compression, A-V impulse systems, passive ankle motion systems and graduated compression stockings are an additional, effective prophylaxis without side effects.

[Transient osteoporosis and osteonecrosis of the femoral head. Risk factors, classification and differential diagnosis]. / Transiente Osteoporose und aseptische Femurkopfnekrose. Risikofaktoren, Klassifikation, Differentialdiagnose.

Osteonecrosis of the femoral head usually affects patients in their third to fifth decade of life. Common risk factors are alcohol, nicotine, corticosteroids, hyperlipidaemia and hypercoagulability. Depending on the stage of the osteonecrosis, the diagnosis is confirmed by radiographs, magnetic resonance imaging or scintigraphy. The ARCO classification (Association Research Circulation Osseous), which is based on older classifications recommended by Ficat/Arlet, Steinberg, Koo or Marcus/Enneking, is a valuable prognostic tool for finding an adequate treatment option. Transient osteoporosis of the hip is controversially discussed as a pre-stage of osteonecrosis or a self-limiting condition based on reflex dystrophy. Conservative and operative treatment options are reported in the literature. Recently published data favour core decompression as an effective procedure for early stage osteonecrosis and transient osteoporosis.

WITHDRAWN: Immunohistochemical localization of Ih channel HCN3 in the rat brain.

Ahead of Print article withdrawn by publisher

Proposal for a histopathological consensus classification of the periprosthetic interface membrane.

AIMS: The introduction of clearly defined histopathological criteria for a standardised evaluation of the periprosthetic membrane, which can appear in cases of total joint arthroplasty revision surgery. METHODS: Based on histomorphological criteria, four types of periprosthetic membrane were defined: wear particle induced type (detection of foreign body particles; macrophages and multinucleated giant cells occupy at least 20% of the area; type I); infectious type (granulation tissue with neutrophilic granulocytes, plasma cells and few, if any, wear particles; type II); combined type (aspects of type I and type II occur simultaneously; type III); and indeterminate type (neither criteria for type I nor type II are fulfilled; type IV). The periprosthetic membranes of 370 patients (217 women, 153 men; mean age 67.6 years, mean period until revision surgery 7.4 years) were analysed according to the defined criteria. RESULTS: Frequency of histopathological membrane types was: type I 54.3%, type II 19.7%, type III 5.4%, type IV 15.4%, and not assessable 5.1%. The mean period between primary arthroplasty and revision surgery was 10.1 years for type I, 3.2 years for type II, 4.5 years for type III and 5.4 years for type IV. The correlation between histopathological and microbiological diagnosis was high (89.7%), and the inter-observer reproducibility sufficient (85%). CONCLUSION: The classification proposed enables standardised typing of periprosthetic membranes and may serve as a tool for further research on the pathogenesis of the loosening of total joint replacement. The study highlights the importance of non-infectious, non-particle induced loosening of prosthetic devices in orthopaedic surgery (membrane type IV), which was observed in 15.4% of patients.

Simultaneous, phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody enhances antiangiogenic effects in vivo.

BACKGROUND: Intracellular antibodies (intrabodies) have been used for the generation of phenotypic knockouts in vivo by surface depletion of extracellular or transmembrane proteins. Intrabodies present an alternative to methods of gene inactivation that target genomic DNA or m-RNA, such as RNA interference. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects in tumor growth. METHODS: In order to generate a precise tool for the simultaneous silencing of two independent signaling pathways essential for angiogenesis, we developed a bispecific, tetravalent endoplasmatic reticulum (ER)-targeted intradiabody, against Tie-2 and VEGF-R2. RESULTS: Using an adenovirus mediated gene delivery system, we achieved the simultaneous downregulation of the two cell surface receptors and demonstrate that the intradiabody is significantly more powerful with respect to efficiency and duration of surface depletion of Tie-2 and VEGF-R2 when compared to scFv intrabodies. In a human melanoma xenograft mouse model, we could show that blockade of both VEGF-R2 and Tie-2 pathways or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2 % and 74.4 %). CONCLUSION: We demonstrate for the first time that simultaneous inhibition of the VEGF and the Tie-2 receptor pathways result in additive antiangiogenic effects in vitro and in vivo as compared to single VEGF receptor pathway blockade, strengthening the potential of simultaneous targeting of multiple pathways as a therapeutic strategy.

[Long-term results of uncemented Parhofer-Mönch screw rings]. / Langzeitergebnisse der zementfreien Parhofer-Mönch Schraubpfanne.

AIM: The aim of this study was an analysis of the long-term behaviour and implant migration of the Parhofer-Mönch-screw cup seen in patients between 1982 and 1991. METHOD: 92 cups (m : f = 53 : 39, mean age 53 +/- 7 years) were included mainly prospectively. After 118 +/- 45 months all patients were examined clinically and radiologically. Digital migration analysis was performed using the single-film X-ray analysis (Einbildröntgenanalyse, EBRA). RESULTS: 5 patients had died. 32 cups were revised, in 7 patients a loosening of the cup was suspected. The 10-year-survival was 71.4 %. In 53 of 81 analysed cups a migration of more than 1 mm was shown, 28 cups did not migrate. In comparison to these stable implants the survival of migrated cups was significantly inferior. CONCLUSION: The 10-year-survival and the high rate of implant migration document the poor results of the PM cup. In spite of an extraordinary primary stability, the failure of secondary osseointegration represents the main cause of loosening in this type of cup.

Magnetic resonance imaging criteria of successful core decompression in avascular necrosis of the hip.

To identify imaging criteria that determine the outcome of core decompression (CD) in femoral-head avascular necrosis (AVN). Radiographs and magnetic resonance imaging (MRI) of 65 hips with early stage AVN treated by core decompression between January 1990 and December 2000 for AVN were reviewed. All hips were categorized into two groups according to the result of CD using total hip arthroplasty (THA) as an end point. Hips that had no THA at follow-up were allocated to group I; those treated with a THA were allocated to group II. CD results were calculated for each group using THA as an end point. The parameters analyzed were the presence or absence of edema associated with the double-line sign on the preoperative MRI, the type of epiphyseal scar (ES) according to Jing, and the type of necrosis according to Mitchell. On follow-up, 45 hips had no THA (group I); 20 patients had a THA (group II). Patients with a radiographic crescent sign and those with edema associated with the double-line sign progressed to THA significantly more frequently. The extent of the necrosis had less discriminatory effect between the two groups. ES and necrotic tissue types had no prognostic value. In regard to the success of CD, it is important to differentiate on MRI between a double line sign plus bone marrow edema and a double-line sign only.

Treatment of transient bone marrow oedema of the hip--a comparative study.

Between 1990 and 2000, we treated 43 patients with transient bone marrow oedema of the hip. Five were treated with nonsteroidal antiinflammatory drugs (NSAIDs) and limited weight bearing, and 38 by core decompression followed by limited weight bearing. At follow-up 2-10 years later, all patients were assessed by a structured interview as well as the Harris hip score (HHS) and the Western Ontario and MacMaster Universities Osteoarthritis Index (WOMAC). Both groups reached the same clinical outcome (HHS and WOMAC). Core decompression enabled a significantly faster recovery. There were no complications, but progression to avascular necrosis was seen in both groups. Core decompression induced fast pain relief, making it the preferable treatment.

Transient marrow edema syndrome of the hip: results after core decompression. A prospective MRI-controlled study in 22 patients.

BACKGROUND: Transient marrow edema syndrome (TMES) of the hip is a disease of acute onset and severe functional disability. There is histological evidence for an ischemic etiology of TMES of the hip. Core decompression as applied for avascular necrosis (AVN) of the hip is therefore a therapeutic alternative to conservative therapy, the latter leading only to a reduction of symptoms but never a shortening of the course of the disease. METHODS: Between January 1998 and June 2000, 22 hips with TMES were treated with core decompression in our department. TMES was diagnosed by exclusion. MRI was done preoperatively and at 6 months postoperatively. The postoperative MRI result was classified into three categories. RESULTS: After an average of 7.2 (range 1-30) days, all patients were pain-free after core decompression. In 2 patients, TMES progressed to AVN despite core decompression. All others had no signal alterations of the head of the femur on MRI after 6 months. The postoperative Harris Hip Score (HHS) in patients with TMES was on average 93.7 (range 77-95); in patients with AVN, the postoperative HHS was 47 (range 45-49). CONCLUSION: Our results demonstrate that core decompression of the hip significantly shortens the natural course of disease of TMES of the hip.

The contactin-related protein FAR-2 defines purkinje cell clusters and labels subpopulations of climbing fibers in the developing cerebellum.

FAR-2 is a novel neural member of the Ig superfamily, which is related to F11/F3/contactin and axonin-1/TAG-1. This protein is expressed by subpopulations of Purkinje cells in the chicken cerebellum and FAR-2-positive clusters of these neurons alternate with FAR-2-negative clusters in both tangential dimensions of the cerebellar cortex. Furthermore, FAR-2 is also expressed by one type of Purkinje cell afferents, namely, the climbing fibers, and different subpopulations of these axons show distinct levels of FAR-2 expression. Homology modeling using axonin-1 as a template reveals that the four aminoterminal Ig domains of FAR-2 form a compact U-shaped structure, which is likely to contain functionally important ligand-binding sites. FAR-2 is binding to the Ig superfamily protein NgCAM/L1, but not to the related receptor NrCAM, and it is also interacting with the modular ECM protein tenascin-R. These results suggest that FAR-2 may contribute to the formation of somatotopic maps of cerebellar afferents during the development of the nervous system.

In vivo activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy.

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 10(2)-fold less toxic than etoposide itself in in vitro assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation in vivo was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm3 s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.