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Cytokines and chemokines not up-regulated by NB-MSC interaction and lack of inhibition of STAT3 activation by anti-IL-6/IL-6R

Supplementary Tables

Purpose: Medulloblastoma in children can be categorized into at least four molecular subgroups, offering the potential for targeted therapeutic approaches to reduce treatment-related morbidities. Little is known about the role of tumor microenvironment in medulloblastoma or its contribution to these molecular subgroups. Tumor microenvironment has been shown to be an important source for therapeutic targets in both adult and pediatric neoplasms. In this study, we investigated the hypothesis that expression of genes related to tumor-associated macrophages (TAM) correlates with the medulloblastoma molecular subgroups and contributes to a diagnostic signature.Methods: Gene-expression profiling using human exon array (n = 168) was analyzed to identify medulloblastoma molecular subgroups and expression of inflammation-related genes. Expression of 45 tumor-related and inflammation-related genes was analyzed in 83 medulloblastoma samples to build a gene signature predictive of molecular subgroups. TAMs in medulloblastomas (n = 54) comprising the four molecular subgroups were assessed by immunohistochemistry (IHC).Results: A 31-gene medulloblastoma subgroup classification score inclusive of TAM-related genes (CD163 and CSF1R) was developed with a misclassification rate of 2%. Tumors in the Sonic Hedgehog (SHH) subgroup had increased expression of inflammation-related genes and significantly higher infiltration of TAMs than tumors in the Group 3 or Group 4 subgroups (P < 0.0001 and P < 0.0001, respectively). IHC data revealed a strong association between location of TAMs and proliferating tumor cells.Conclusions: These data show that SHH tumors have a unique tumor microenvironment among medulloblastoma subgroups. The interactions of TAMs and SHH medulloblastoma cells may contribute to tumor growth revealing TAMs as a potential therapeutic target. Clin Cancer Res; 21(6); 1457–65. ©2014 AACR.

Supplemental Figure 1 and Figure 2 Legends Supplemental Tables 1-5 Legends

A: List of antibodies used in the study B: Clinical characteristics of patients from which CAF-MSC and BM-MSC were isolated.

PDF file 92K, Supplemental Fig 5: Expression of PID1 protein in human brain tumors and cell lines

Permission letter regarding inclusion of information of CHLA-255 C-Myc over-expression

Purpose:We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design:The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results:ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions:Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

Supplemental Table 3. Sociodemographic characteristics of the California Breast Cancer Survivorship Consortium by pre-diagnosis BMI.

Purpose: Immunotherapy of high-risk neuroblastoma using the anti-GD2 antibody dinutuximab induces antibody-dependent cell-mediated cytotoxicity (ADCC). Galunisertib, an inhibitor of TGFβR1, 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 TGFβ1-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 TGFβ1 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 TGFβ1, 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 AACR.See related commentary by Zenarruzabeitia et al., p. 615

PDF file - 190K, Supplemental Fig 2: Proneural and Neural GBMs have higher PID1 mRNA; higher PID1 mRNA correlates with higher OS in patients with gliomas.

Supplemental Methods and Supplemental Figures 1-9 Supplemental Figure 1. Overview of samples studies and 31-gene signature development Supplemental Figure. 2. Non-negative matrix factorization (NMF) analysis of the combined cohort of medulloblastoma samples (n=168) (A) Cophenetic correlation coefficient based on 2000 clustering runs for 2-10 clusters utilizing genes obtained at varying coefficients of variation; the data demonstrate that the highest cophenetic correlation coefficient was obtained at 4 clusters with 369 genes. (B) NMF consensus heatmap of 4 subgroups using the dataset containing 369 genes with high coefficient of variation. (C) Silhouette plot for identification of outliers. Outliers (n=31) were defined as samples with a silhouette width

CAF-MSC and BM-MSC enhance cell viability of NB cells in vitro and protect tumor cells from drug-induced apoptosis in vitro.

Supplementary Figure Legends for A1-A3

Supplementary Figure S2 shows the results of intracellular staining of aNK cells for perforin and granzyme A, as requested by reviewer #3.

PDF file - 159K, Supplemental Fig 1: PID1 mRNA is lower in less favorable medulloblastomas in two additional independent datasets; PID1 mRNA correlates with rf-PFS when analyzing CHLA medulloblastomas by tertiles.

PDF file - 17K, Supplemental Fig 4: PID1 increases the proportion of cells in Sub G0/G1 in CHLA-259 medulloblastoma cells.

Purpose: We present here the first report of PID1 (Phosphotyrosine Interaction Domain containing 1; NYGGF4) in cancer. PID1 was identified in 2006 as a gene that modulates insulin signaling and mitochondrial function in adipocytes and muscle cells.Experimental Design and Results: Using four independent medulloblastoma datasets, we show that mean PID1 mRNA levels were lower in unfavorable medulloblastomas (groups 3 and 4, and anaplastic histology) compared with favorable medulloblastomas (SHH and WNT groups, and desmoplastic/nodular histology) and with fetal cerebellum. In two large independent glioma datasets, PID1 mRNA was lower in glioblastomas (GBM), the most malignant gliomas, compared with other astrocytomas, oligodendrogliomas and nontumor brains. Neural and proneural GBM subtypes had higher PID1 mRNA compared with classical and mesenchymal GBM. Importantly, overall survival and radiation-free progression-free survival were longer in medulloblastoma patients whose tumors had higher PID1 mRNA (univariate and multivariate analyses). Higher PID1 mRNA also correlated with longer overall survival in patients with glioma and GBM. In cell culture, overexpression of PID1 inhibited colony formation in medulloblastoma, atypical teratoid rhabdoid tumor (ATRT), and GBM cell lines. Increasing PID1 also increased cell death and apoptosis, inhibited proliferation, induced mitochondrial depolaization, and decreased serum-mediated phosphorylation of AKT and ERK in medulloblastoma, ATRT, and/or GBM cell lines, whereas siRNA to PID1 diminished mitochondrial depolarization.Conclusions: These data are the first to link PID1 to cancer and suggest that PID1 may have a tumor inhibitory function in these pediatric and adult brain tumors. Clin Cancer Res; 20(4); 827–36. ©2013 AACR.

Table A1. Therapies received for samples submitted on this study; Table A2. Genes and Primer and Probe Sets for NB5 assay; Table A3. Clinical Evaluations and NB5 TLDA Assays Performed; Table A4. Contribution of Individual Gene to NB5 Signature and AUC.

Supplemental Table 1: Complete CBCSC cohort description, by all baseline model variables (N=12,098). Supplemental Table 2: Project specific sub-cohort description. Supplemental Table 3: Hazard ratio (HR) estimates and 95% confidence intervals from Cox regression analysis of breast cancer-specific mortality, for the racial/ethnic groups only model vs the full baseline model. (AJCC Stage I-II cases only). Supplemental Table 4: Hazard ratio (HR) estimates and 95% confidence intervals from Cox regression analysis of breast cancer-specific mortality, for the full baseline model vs the baseline model + specific domain variables, for the subsets of cases for which domain data were available(AJCC stage III cases Only). Supplemental Figure 1: Same as Figure 1, but with analysis restricted to AJCC stage I-II cases only.

Targeting CD105+ Microenvironment cells enhances immunotherpy of Dinutuxmab with aNK cells against neuroblastoma

CAF-MSC and BM-MSC induce activation of STAT3 and ERK1/2 signaling in NB cells

PDF file - 440K, Supplemental Table 1: Characteristics of the CHLA Medulloblastoma Patients. Supplemental Table 2: Neural and Proneural GBM subgroups have higher PID1 mRNA compared to Classical and Mesenchymal.

Supplementary Table S1 is a list of antibodies used in the study. It has been updated to include antibodies used in experiments requested by reviewer #3.

Supplemental Figure 1 and Figure 2 Supplemental Tables 1-5

Supplemental Tables 1-6 Supplemental Table 1. Patient and tumor characteristics Supplemental Table 2. TLDA design (45 genes) Supplemental Table 3. Information for samples evaluated using HuE Supplemental Table 4. TLDA 31-gene molecular classification of core samples Supplemental Table 5. TLDA 31-gene classification for samples without HuEx data Supplemental Table 6. TLDA 31-gene Confusion Matrix

CAF-MSC increase tumor engraftment and growth in xenograft model.

Purpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).Experimental Design: Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma.Results: Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56+CD3−CD14− NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151–9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added.Conclusion: Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18. Clin Cancer Res; 19(8); 2132–43. ©2013 AACR.

Comparison of 1 year EFS in two groups defined by a range of ∆CT cut points (left column) and corresponding ROC analysis of the ability of ∆CT values above the cutpoint to predict 1 year EFS (right column), for assays based on different combinations of genes. NB5 assay (A, B), Phox2B/TH/DDC (C, D), Phox2B/TH (E, F), Phox2B/TH/Dcx (G, H), Th/Dcx (I, J). These analyses show that the NB5 assay is at least as or more predictive then the other gene combinations.

PDF file - 1549K, Supplemental Figure 1. Cytokine and chemokine release from K562 Clone 9.mbIL21 aAPC-expanded effector cells after 24-hour incubation with neuroblastoma cell line CHLA-255-Fluc alone or with anti-GD2 antibody ch14.18. Supplemental Figure 2. Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved effector cells from a normal donor PBMC that were thawed and cultured for 3 days or thawed and immediately injected intravenously into NOD/SCID mice beginning 7 days after they had received 106 CHLA-255-Fluc neuroblastoma cells intravenously. Supplemental Figure 3. Anti-tumor activity of dose-dense treatment with K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells derived from a patient�s PBMC. Supplemental Figure 4. Anti-tumor activity of K562 Clone 9.mbIL21 aAPC-expanded and cryopreserved aNK cells derived from a patient�s PBMC when treatment was begun 21 days (late treatment) after tumor cell injection.

Cancer-associated fibroblasts (CAF) have been suggested to originate from mesenchymal stromal cells (MSC), but their relationship with MSCs is not clear. Here, we have isolated from primary human neuroblastoma tumors a population of αFAP- and FSP-1–expressing CAFs that share phenotypic and functional characteristics with bone marrow–derived MSCs (BM-MSC). Analysis of human neuroblastoma tumors also confirmed the presence of αFAP- and FSP-1–positive cells in the tumor stroma, and their presence correlated with that of M2 tumor-associated macrophages. These cells (designated CAF-MSCs) enhanced in vitro neuroblastoma cell proliferation, survival, and resistance to chemotherapy and stimulated neuroblastoma tumor engraftment and growth in immunodeficient mice, indicating an effect independent of the immune system. The protumorigenic activity of MSCs in vitro and in xenografted mice was dependent on the coactivation of JAK2/STAT3 and MEK/ERK1/2 in neuroblastoma cells. In a mouse model of orthotopically implanted neuroblastoma cells, inhibition of JAK2/STAT3 and MEK/ERK/1/2 by ruxolitinib and trametinib potentiated tumor response to etoposide and increased overall survival. These data point to a new type of protumorigenic CAF in the tumor microenvironment of neuroblastoma and to STAT3 and ERK1/2 as mediators of their activity. Cancer Res; 77(18); 5142–57. ©2017 AACR.

Addition methods describing construction of the cohort, data harmonization, and statistical details

Supplemental Table 4. Clinicopathologic and treatment characteristics of the California Breast Cancer Survivorship Consortium by pre-diagnosis BMI.

Supplemental Table 1. Neighborhood characteristics of the California Breast Cancer Survivorship Consortium by pre-diagnosis BMI.

Little is known about neighborhood attributes that may influence opportunities for healthy eating and physical activity in relation to breast cancer mortality. We used data from the California Breast Cancer Survivorship Consortium and the California Neighborhoods Data System (CNDS) to examine the neighborhood environment, body mass index, and mortality after breast cancer. We studied 8,995 African American, Asian American, Latina, and non-Latina white women with breast cancer. Residential addresses were linked to the CNDS to characterize neighborhoods. We used multinomial logistic regression to evaluate the associations between neighborhood factors and obesity and Cox proportional hazards regression to examine associations between neighborhood factors and mortality. For Latinas, obesity was associated with more neighborhood crowding [quartile 4 (Q4) vs. Q1: OR, 3.24; 95% confidence interval (CI), 1.50–7.00]; breast cancer–specific mortality was inversely associated with neighborhood businesses (Q4 vs. Q1: HR, 0.46; 95% CI, 0.25–0.85) and positively associated with multifamily housing (Q3 vs. Q1: HR, 1.98; 95% CI, 1.20–3.26). For non-Latina whites, lower neighborhood socioeconomic status (SES) was associated with obesity [quintile 1 (Q1) vs. Q5: OR, 2.52; 95% CI, 1.31–4.84], breast cancer–specific (Q1 vs. Q5: HR, 2.75; 95% CI, 1.47–5.12), and all-cause (Q1 vs. Q5: HR, 1.75; 95% CI, 1.17–2.62) mortality. For Asian Americans, no associations were seen. For African Americans, lower neighborhood SES was associated with lower mortality in a nonlinear fashion. Attributes of the neighborhood environment were associated with obesity and mortality following breast cancer diagnosis, but these associations differed across racial/ethnic groups. Cancer Epidemiol Biomarkers Prev; 24(8); 1282–90. ©2015 AACR.

Background: Racial/ethnic disparity in breast cancer–specific mortality in the United States is well documented. We examined whether accounting for racial/ethnic differences in the prevalence of clinical, patient, and lifestyle and contextual factors that are associated with breast cancer–specific mortality can explain this disparity.Methods: The California Breast Cancer Survivorship Consortium combined interview data from six California-based breast cancer studies with cancer registry data to create a large, racially diverse cohort of women with primary invasive breast cancer. We examined the contribution of variables in a previously reported Cox regression baseline model plus additional contextual, physical activity, body size, and comorbidity variables to the racial/ethnic disparity in breast cancer–specific mortality.Results: The cohort comprised 12,098 women. Fifty-four percent were non-Latina Whites, 17% African Americans, 17% Latinas, and 12% Asian Americans. In a model adjusting only for age and study, breast cancer–specific HRs relative to Whites were 1.69 (95% CI, 1.46–1.96), 1.00 (0.84–1.19), and 0.52 (0.33–0.85) for African Americans, Latinas, and Asian Americans, respectively. Adjusting for baseline-model variables decreased disparity primarily by reducing the HR for African Americans to 1.13 (0.96–1.33). The most influential variables were related to disease characteristics, neighborhood socioeconomic status, and smoking status at diagnosis. Other variables had negligible impact on disparity.Conclusions: Although contextual, physical activity, body size, and comorbidity variables may influence breast cancer–specific mortality, they do not explain racial/ethnic mortality disparity.Impact: Other factors besides those investigated here may explain the existing racial/ethnic disparity in mortality. Cancer Epidemiol Biomarkers Prev; 25(7); 1064–72. ©2016 AACR.

PDF file - 89K, Supplemental Table 1. List of antibodies used in this study. Supplemental table 2. Cytokines/chemokines excluded from the Luminex� panels.

PDF file - 42K, Supplemental Fig 3: PID1 also inhibits colony formation using two other plasmid expression vectors.

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.

Combination of ruxolitinib and trametinib inhibits the growth of subcutaneous NB tumors, cell proliferation and STAT3 and ERK1/2 activation in vivo.

Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and immunoglobulin (Ig) class switch recombination in germinal center (GC) B cells. Occasionally, AID can target non-Ig genes and thereby promote GC B-cell lymphomagenesis. We recently showed that the oncogenic BCR-ABL1 kinase induces aberrant expression of AID in pre-B acute lymphoblastic leukemia (ALL) and lymphoid chronic myelogenous leukemia blast crisis. To elucidate the biological significance of aberrant AID expression, we studied loss of AID function in a murine model of BCR-ABL1 ALL. Mice transplanted with BCR-ABL1–transduced AID−/− bone marrow had prolonged survival compared with mice transplanted with leukemia cells generated from AID+/+ bone marrow. Consistent with a causative role of AID in genetic instability, AID−/− leukemia had a lower frequency of amplifications and deletions and a lower frequency of mutations in non-Ig genes, including Pax5 and Rhoh compared with AID+/+ leukemias. AID−/− and AID+/+ ALL cells showed a markedly distinct gene expression pattern, and AID−/− ALL cells failed to downregulate a number of tumor-suppressor genes including Rhoh, Cdkn1a (p21), and Blnk (SLP65). We conclude that AID accelerates clonal evolution in BCR-ABL1 ALL by enhancing genetic instability and aberrant somatic hypermutation, and by negative regulation of tumor-suppressor genes. Cancer Res; 70(19); 7411–20. ©2010 AACR.

Heparan sulfate proteoglycans (HSPG) play a critical role in the interaction of tumor cells and their microenvironment. HSPG activity is dictated by sulfation patterns controlled by sulfotransferases, which add sulfate groups, and sulfatases (Sulf), which remove 6-O-sulfates. Here, we report altered expression of these enzymes in human neuroblastoma cells with higher levels of Sulf-2 expression, a specific feature of MYCN-amplified cells (MYCN-A cells) that represent a particularly aggressive subclass. Sulf-2 overexpression in neuroblastoma cells lacking MYCN amplification (MYCN-NA cells) increased their in vitro survival. Mechanistic investigations revealed evidence of a link between Sulf-2 expression and MYCN pathogenicity in vitro and in vivo. Analysis of Sulf-2 protein expression in 65 human neuroblastoma tumors demonstrated a higher level of Sulf-2 expression in MYCN-A tumors than in MYCN-NA tumors. In two different patient cohorts, we confirmed the association in expression patterns of Sulf-2 and MYCN and determined that Sulf-2 overexpression predicted poor outcomes in a nonindependent manner with MYCN. Our findings define Sulf-2 as a novel positive regulator of neuroblastoma pathogenicity that contributes to MYCN oncogenicity. Cancer Res; 74(21); 5999–6009. ©2014 AACR.