Continuous and bolus intraventricular topotecan prolong survival in a mouse model of leptomeningeal medulloblastoma.

Leptomeningeal metastasis remains a difficult clinical challenge. Some success has been achieved by direct administration of therapeutics into the cerebrospinal fluid (CSF) circumventing limitations imposed by the blood brain barrier. Here we investigated continuous infusion versus bolus injection of therapy into the CSF in a preclinical model of human Group 3 medulloblastoma, the molecular subgroup with the highest incidence of leptomeningeal disease. Initial tests of selected Group 3 human medulloblastoma cell lines in culture showed that D283 Med and D425 Med were resistant to cytosine arabinoside and methotrexate. D283 Med cells were also resistant to topotecan, whereas 1 µM topotecan killed over 99% of D425 Med cells. We therefore introduced D425 Med cells, modified to express firefly luciferase, into the CSF of immunodeficient mice. Mice were then treated with topotecan or saline in five groups: continuous intraventricular (IVT) topotecan via osmotic pump (5.28 µg/day), daily bolus IVT topotecan injections with a similar daily dose (6 µg/day), systemic intraperitoneal injections of a higher daily dose of topotecan (15 µg/day), daily IVT pumped saline and daily intraperitoneal injections of saline. Bioluminescence analyses revealed that both IVT topotecan treatments effectively slowed leptomeningeal tumor growth in the brains. Histological analysis showed that they were associated with localized brain necrosis, possibly due to backtracking of topotecan around the catheter. In the spines, bolus IVT topotecan showed a trend towards slower tumor growth compared to continuous (pump) IVT topotecan, as measured by bioluminescence. Both continuous and bolus topotecan IVT showed longer survival compared to other groups. Thus, both direct IVT topotecan CSF delivery methods produced better anti-medulloblastoma effect compared to systemic therapy at the dosages used here.

Expression of Five Neuroblastoma Genes in Bone Marrow or Blood of Patients with Relapsed/Refractory Neuroblastoma Provides a New Biomarker for Disease and Prognosis.

Purpose: We determined whether quantifying neuroblastoma-associated mRNAs (NB-mRNAs) in bone marrow and blood improves assessment of disease and prediction of disease progression in patients with relapsed/refractory neuroblastoma.Experimental Design: mRNA for CHGA, DCX, DDC, PHOX2B, and TH was quantified in bone marrow and blood from 101 patients concurrently with clinical disease evaluations. Correlation between NB-mRNA (delta cycle threshold, ΔCt, for the geometric mean of genes from the TaqMan Low Density Array NB5 assay) and morphologically defined tumor cell percentage in bone marrow, 123I-meta-iodobenzylguanidine (MIBG) Curie score, and CT/MRI-defined tumor longest diameter was determined. Time-dependent covariate Cox regression was used to analyze the relationship between ΔCt and progression-free survival (PFS).Results: NB-mRNA was detectable in 83% of bone marrow (185/223) and 63% (89/142) of blood specimens, and their ΔCt values were correlated (Spearman r = 0.67, P < 0.0001), although bone marrow Ct was 7.9 ± 0.5 Ct stronger than blood Ct When bone marrow morphology, MIBG, or CT/MRI were positive, NB-mRNA was detected in 99% (99/100), 88% (100/113), and 81% (82/101) of bone marrow samples. When all three were negative, NB-mRNA was detected in 55% (11/20) of bone marrow samples. Bone marrow NB-mRNA correlated with bone marrow morphology or MIBG positivity (P < 0.0001 and P = 0.007). Bone marrow and blood ΔCt values correlated with PFS (P < 0.001; P = 0.001) even when bone marrow was morphologically negative (P = 0.001; P = 0.014). Multivariate analysis showed that bone marrow and blood ΔCt values were associated with PFS independently of clinical disease and MYCN gene status (P < 0.001; P = 0.055).Conclusions: This five-gene NB5 assay for NB-mRNA improves definition of disease status and correlates independently with PFS in relapsed/refractory neuroblastoma. Clin Cancer Res; 23(18); 5374-83. ©2017 AACR.

TGFßR1 Blockade with Galunisertib (LY2157299) Enhances Anti-Neuroblastoma Activity of the Anti-GD2 Antibody Dinutuximab (ch14.18) with Natural Killer Cells.

PURPOSE: Immunotherapy of high-risk neuroblastoma using the anti-GD2 antibody dinutuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC). Galunisertib, an inhibitor of TGFßR1, was examined for its ability to enhance the efficacy of dinutuximab in combination with human ex vivo activated NK (aNK) cells against neuroblastoma. EXPERIMENTAL DESIGN: TGFB1 and TGFBR1 mRNA expression was determined for 249 primary neuroblastoma tumors by microarray analysis. The ability of galunisertib to inhibit SMAD activity induced by neuroblastoma patient blood and bone marrow plasmas in neuroblastoma cells was tested. The impact of galunisertib on TGFß1-induced inhibition of aNK cytotoxicity and ADCC in vitro and on anti-neuroblastoma activity in NOD-scid gamma (NSG) mice was determined. RESULTS: Neuroblastomas express TGFB1 and TGFBR1 mRNA. Galunisertib suppressed SMAD activation in neuroblastoma cells induced by exogenous TGFß1 or by patient blood and bone marrow plasma, and suppressed SMAD2 phosphorylation in human neuroblastoma cells growing in NSG mice. In NK cells treated in vitro with exogenous TGFß1, galunisertib suppressed SMAD2 phosphorylation and restored the expression of DNAM-1, NKp30, and NKG2D cytotoxicity receptors and the TRAIL death ligand, the release of perforin and granzyme A, and the direct cytotoxicity and ADCC of aNK cells against neuroblastoma cells. Addition of galunisertib to adoptive cell therapy with aNK cells plus dinutuximab reduced tumor growth and increased survival of mice injected with two neuroblastoma cell lines or a patient-derived xenograft. CONCLUSIONS: Galunisertib suppresses activation of SMAD2 in neuroblastomas and aNK cells, restores NK cytotoxic mechanisms, and increases the efficacy of dinutuximab with aNK cells against neuroblastoma tumors. Clin Cancer Res; 23(3); 804-13. ©2016 AACRSee related commentary by Zenarruzabeitia et al., p. 615.

The clinical management and outcomes of cervical neuroblastic tumors.

BACKGROUND: Although patients with peripheral neuroblastoma (NB; pelvic and thoracic) typically have better outcomes and less aggressive disease compared with patients with abdominal disease, little has been published with regard to the management and outcomes of patients with cervical NB. Herein, we sought to determine the characteristics of cervical neuroblastic tumors and the effect of extent of resection on survival and outcomes. METHODS: We performed a retrospective review of 325 children with neuroblastic tumors at Children's Hospital Los Angeles over a 15-y period (January 1990-February 2015). Data collected from the medical record included location of tumor, age at diagnosis, age at resection, extent of resection, chemotherapy course, International Neuroblastoma Staging System stage, histologic International Neuroblastoma Pathology Classification, and MYCN amplification, a poor prognostic marker. Outcome variables included postoperative complications and overall survival. RESULTS: Twelve patients (3.6%) were found to have cervical neuroblastic tumors (nine NBs, one ganglioneuroblastoma, and two ganglioneuromas). All had favorable histology, and none (0/12) had MYCN amplification. Of the NB patients, four of nine patients underwent resection, whereas the other five underwent biopsy followed by chemotherapy or observation alone. Of the 12 total patients, six underwent gross total resection, four (67%) of which developed complications. At a median follow-up of 4.4 y, there were no recurrences or deaths. CONCLUSIONS: Cervical neuroblastic tumors represent favorable lesions with good outcomes similar to other peripheral neuroblastic tumors. In our study, survival was excellent regardless of extent of tumor resection. Based on our data, we recommend a minimally aggressive surgical approach in managing children with cervical neuroblastic tumors.

Oxaliplatin and Doxorubicin for relapsed or refractory high-risk neuroblastoma.

Patients with relapsed or refractory neuroblastoma have poor long-term survival. New therapeutic regimens are needed. Doxorubicin and cisplatin are commonly used in the treatment of high-risk neuroblastoma. Oxaliplatin, a platinum compound with a 1,2-diaminocyclohexan carrier ligand, is more potent than cisplatin with less nephrotoxicity and ototoxicity. We treated seven relapsed/refractory neuroblastoma patients using oxaliplatin (105-130 mg/m(2)) and doxorubicin (60-75 mg/m(2)) together with dexrazoxane (10 mg/mg of doxorubicin) administered intravenously every three weeks. Prolonged thrombocytopenia causing treatment delay was observed when oxaliplatin was administered at 130 mg/m(2). A reduced dose of oxaliplatin 105 mg/m(2) on day 1 with doxorubicin at 20 mg/m(2)/dose on days 1-3 was well tolerated. Sensory neuropathies were mild and transient. No cardiotoxicity was noted despite all patients having a history of prior anthracycline exposure. Best responses included 1 complete response, 1 partial response, 1 mixed response, 3 stable diseases. In our cohort of heavily pretreated relapsed and refractory neuroblastoma patients, the combination of oxaliplatin and doxorubicin demonstrated anti-tumor activity and merits further investigation.

Pilot study assessing a seven-day continuous intrathecal topotecan infusion for recurrent or progressive leptomeningeal metastatic cancer.

OBJECTIVE: To determine the feasibility and toxicity profile of topotecan administered as a seven-day continuous intrathecal infusion for patients with leptomeningeal metastasis secondary to recurrent or progressive central nervous system cancer. STUDY DESIGN: Two patients with central nervous system leptomeningeal metastasis were treated with a seven-day continuous infusion of topotecan (0.2 mg/day) administered via continuous intrathecal/intraventricular infusion at a rate of 0.6 mL/h, totaling 1.4 mg/course. CSF and plasma concentrations of topotecan closed lactone (the active metabolite) were quantified at various points during topotecan infusion. Patients were monitored for neurologic and systemic toxicities according to NCI common toxicity criteria. RESULTS: Both patients tolerated the seven-day continuous topotecan without any significant adverse events. One patient received a second course 21 days after treatment initiation. CSF concentration of topotecan closed lactone ranged from 3.73 to 312 ng/mL (median = 131 ng/mL) and plasma topotecan closed lactone ranged from 0.44 to 1.78 ng/mL (median = 0.92 ng/mL). The median CSF topotecan concentration was greater than the median serum topotecan concentration by a 44-fold magnitude when samples were obtained at the same time point. None of the patients experienced any grade 3 or higher hematological toxicities or signs of arachnoiditis. CONCLUSION: A seven-day continuous intrathecal infusion of topotecan is well tolerated and has the potential of maximizing central nervous system drug exposure.

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFß1.

BACKGROUND: Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFß1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression. METHODS: Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFß1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma. RESULTS: CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFß1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFß1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFß1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide. CONCLUSION: Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFß1.