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Telomeres play important roles in genome stability and cell proliferation. High risk neuroblastoma (HRNB), an aggressive childhood cancer, is especially reliant on telomere maintenance. Three recurrent genetic aberrations in HRNB (MYCN amplification, TERT re-arrangements, and ATRX mutations) are mutually exclusive and each capable of promoting telomere maintenance mechanisms (i.e., through telomerase or ALT). We analyzed a panel of 5 representative HRNB cell lines and 30 HRNB surgical samples using assays that assess average telomere lengths, length distribution patterns, single-stranded DNA on the G- and C-strand, as well as extra-chromosomal circular telomeres. Our analysis pointed to substantial and variable degrees of telomere DNA damage in HRNB, including pervasive oxidative lesions. Moreover, unlike other cancers, neuroblastoma consistently harbored high levels of C-strand ssDNA overhangs and t-circles, which are consistent with active “telomere trimming”. This feature is observed in both telomerase- and ALT-positive tumors and irrespective of telomere length distribution. Moreover, evidence for telomere trimming was detected in normal neural tissues, raising the possibility that TMMs in HRNB evolved in the face of a canonical developmental program of telomere shortening. Telomere trimming by itself appears to distinguish neuroectodermal derived tumors from other human cancers, a distinguishing characteristic with both biologic and therapeutic implications.

Immune checkpoint inhibitors (ICI) have shown great promise in a wide spectrum of adult solid and hematological malignancies, achieving objective tumor responses and prolonging survival. However, there is limited clinical success amongst pediatric patients. In this review, we summarize the current understanding of ICI and present an up-to-date overview of recent and ongoing clinical trials of ICI in pediatric malignancies. In addition, we will discuss immunologic and clinical difficulties in this young population, as well as future prospects for combination of ICI with other immune-based and conventional treatments.

Purpose To evaluate local control after 21-Gy radiation therapy (RT) to the primary site in patients with high-risk neuroblastoma. Methods and Materials After receiving dose-intensive chemotherapy and gross total resection (GTR), 246 patients (aged 1.2-17.9 years, median 4.0 years) with high-risk neuroblastoma underwent RT to the primary site at Memorial Sloan Kettering from 2000 to 2014. Radiation therapy consisted of 21 Gy in twice-daily fractions of 1.5 Gy each. Local failure (LF) was correlated with biologic prognostic factors and clinical findings at the time of diagnosis and start of RT. Results Median follow-up of surviving patients was 6.4 years. Cumulative incidence of LF was 7.1% at 2 years after RT and 9.8% at 5 years after RT. The isolated LF rate was 3.0%. Eighty-six percent of all local failures were within the RT field. Local control was worse in patients who required more than 1 surgical resection to achieve GTR (22.4% vs 8.3%, P =.01). There was also a trend toward inferior local control with MYCN- amplified tumors or serum lactate dehydrogenase ≥1500 U/L ( P =.09 and P =.06, respectively). Conclusion After intensive chemotherapy and maximal surgical debulking, hyperfractionated RT with 21 Gy in high-risk neuroblastoma results in excellent local control. Given the young patient age, concern for late effects, and local control >90%, dose reduction may be appropriate for patients without MYCN amplification who achieve GTR.

Neuroblastoma, a malignancy of multipotent embryonic neural crest cells, is the most common extracranial solid cancer in childhood and most common cancer in infancy. Cellular phenotype has been shown to be an important determinant of the malignant potential in human neuroblastoma cells and tumors. Whereas neuroblastic (N-type) are moderately malignant and nonneuronal (S-type) cells are nonmalignant, I-type stem cells are highly tumorigenic, irrespective of N-myc amplification status. In the present study, we sought to determine which genes were overexpressed in the I-type cells which might characterize and maintain the stem cell state and/or malignancy of human neuroblastoma cancer stem cells. We used a microarray platform to compare the steady-state expression levels of mRNAs from 13 human neuroblastoma cell lines representing the three cellular phenotypes. Using qRT-PCR and Western blot analyses, we identified seven genes whose expression is consistently elevated exclusively in neuroblastoma cancer stem cells: CD133, KIT, NOTCH1, GPRC5C, PIGF2, TRKB, and LNGFR. Moreover, we show that the genes are phenotype specific, as differentiation of I-type BE(2)-C cells to either an N- or S-type morphology results in significantly reduced mRNA expression. Finally, we show that NOTCH1 plays an important role in maintaining the stem cell phenotype. The identification and characterization of these genes, elevated in highly malignant neuroblastoma stem cells, could provide the basis for developing novel therapies for treatment of this lethal childhood cancer.

Background We used a novel regimen for neuroblastoma (NB) that had responded inadequately to standard chemotherapy which now includes topotecan in induction or second-line therapy. Patients and methods We retrospectively studied 38 patients who received one or two courses of high-dose cyclophosphamide (140 mg/kg)-irinotecan (CPT-11) (250 mg/m2)-vincristine (HD-CCV) as treatment for NB that had responded incompletely to induction but had never progressed. Treatment was outpatient and was preceded and followed by extent-of-disease and toxicity evaluations because the patients were being considered for enrolment on formal protocols. Progression-free survival (PFS) was calculated from day 1 of HD-CCV. Results Common toxicities were grade 4 myelosuppression and grade 2 diarrhoea. Responses – 5 complete (CR), 3 partial (PR), 4 mixed (MR) – occurred in 12/28 (43%) patients treated ⩽9 months, and in 1/10 (10%) patients treated >10 months, from diagnosis. HD-CCV was the initial salvage regimen after topotecan-containing induction in 5 patients, achieving 1 CR, 1 MR and 3 stable disease (NR). HD-CCV produced responses (2 PR, 3 MR) in all 5 patients previously treated with CPT-11/ temozolomide. In contrast, all 6 patients treated post-HD-CCV with CPT-11/temozolomide had NR to the latter. Post-HD-CCV treatments included immunotherapy, targeted radiotherapy and/or chemotherapy. PFS was 64% (±8%) at 24 months, with 20 patients progression-free at 2+-to-36+ (median 16+) months and 10 in first CR at 9+-to-36+ (median 16+) months. Conclusions HD-CCV offers a treatment option against topotecan-resistant NB. Results support the concept that combining CPT-11 with very high doses of alkylators can yield greater antitumour effect.

Background/purpose Image-defined vessel encasement is a significant risk factor for surgical complications and incomplete resection for intermediate-risk tumors. We sought to examine the impact of vessel encasement on complications or resectability in intermediate-risk or high-risk patients after neoadjuvant chemotherapy. Methods We retrospectively reviewed 207 consecutive patients with circumferential encasement of the renal vessels, celiac axis, and/or superior mesenteric artery (SMA) who underwent resection between 1991 and 2009. Specifically, we evaluated resection rates, complications, and outcome. Results Median age at diagnosis was 3.0 years, and 79% of patients had stage 4 disease. Of known MYCN status, 23.4% had MYCN amplification. Vessel encasement included renal vessels, celiac axis, or SMA alone in 107, 7, and 4 patients, respectively. Both the renal vessels and celiac axis were encased in 5 patients, renal vessels and SMA in 7 patients, and celiac axis and SMA in 14 patients. Sixty-three patients had all 3 vessels encased. The gross total resection (GTR) rate was 94%. No operative or postoperative deaths occurred. The overall complication rate was 34.8% (n = 72). Overall 5-year survival (± SEM) was 67.4% (± 7.4%). Conclusion Encasement of major visceral arteries in patients with neuroblastoma who have received chemotherapy does not preclude gross total resection.

Background GD2/GM2 synthase is a key enzyme in the synthesis of GD2 and GM2 gangliosides found on the surface of neuroblastoma and small cell lung carcinoma (SCLC) cells. In neuroblastoma, persistent levels of GD2/GM2 synthase RNA in bone marrow (BM) following therapy portend poorer progression-free and overall survival. We conducted this study to determine if GD2/GM2 synthase RNA could be detected in SCLC cell lines and human tissues, and whether mRNA transcript levels corresponded with disease status. Experimental design Initially, a pilot study enrolled patients with SCLC to determine the rate of GD2 expression at various points in the patients’ disease course. Peripheral blood (PB), bone marrow and tumor tissues were used to measure GD2/GM2 synthase levels. In addition, SCLC cell lines were analyzed for GD2/GM2 synthase expression. Based on data from that initial analysis, a prospective trial was developed enrolling patients with newly diagnosed SCLC and following them serially. GD2/GM2 synthase transcript was determined by a sensitive quantitative reverse transcription-PCR (qRT-PCR) assay and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results Six SCLC cell lines were assayed for expression of GD2/GM2 synthase, and high expression was detected in all. GD2/GM2 synthase transcript levels were obtained from tumor tissue, BM, or PB of 29 patients in the pilot study. 6/10 (60%) tumor tissues or BM samples were positive (median 332.7 units; range 13–2323 units); 8/19 (42%) untreated patients were GD2/GM2 synthase positive in their PB prior to beginning therapy (median 10.2; range 5.1–32.2); 3/4 (75%) patients who were first tested when they developed recurrent disease were positive in their PB (median 16.1; range 8.5–19.9). The fourth patient had an initial value of 2.0 (negative), which increased to 8.4 (positive) within 1 month without treatment. Seven of 12 patients with baseline positive GD2/GM2 synthase values had post-treatment levels measured, all of which were ≤5 or showed a >50% decrease following successful treatment. Patients in the prospective trial demonstrated lower rates of positivity, with only 3/26 (12%) patients exhibiting detectable transcript levels in the peripheral blood prior to treatment. All 3 of these patients had their transcript levels fall below 5 after treatment. 11/26 patients had baseline levels of zero. Bone marrow was drawn at baseline on 7 patients in the prospective trial and 3 (43%) had transcript levels above 5 (range 0.65–27.43 units). There was no correlation between elevated levels in the BM and elevated levels in the PB. Conclusions Although initial studies demonstrated that GD2/GM2 synthase transcripts were measurable in the peripheral blood of SCLC patients at diagnosis and declined with successful treatment, in a separate prospective study, these results could not be confirmed. Thus, GD2/GM2 is not a reliable biomarker in SCLC.

Background Many widely used botanical medicines are claimed to be immune enhancers. Clear evidence of augmentation of immune responses in vivo is lacking in most cases. To select botanicals for further study based on immune enhancing activity, we study them here mixed with antigen and injected subcutaneously (s.c.). Globo H and GD3 are cell surface carbohydrates expressed on glycolipids or glycoproteins on the cell surface of many cancers. When conjugated to keyhole limpet hemocyanin (KLH), mixed with an immunological adjuvant and administered s.c. the magnitude of the antibody responses against globo H, GD3 and KLH depend largely on the potency of the adjuvant. We describe here the results obtained using this s.c. immunization model with seven botanicals purported to have immune stimulant effects. Methods Groups of 5–10 mice were immunized with globo H–KLH or GD3–KLH mixed with botanical, saline or positive control immunological adjuvant, s.c. three times at 1 week intervals. Antibody responses were measured 1 and 2 weeks after the 3rd immunization. The following seven botanicals and fractions were tested: (1) H-48 (Honso USA Co.), (2) Coriolus versicolor raw water extract, purified polysaccharide-K (PSK) or purified polysaccharide-peptide (PSP) (Institute of Chinese Medicine (ICM)), (3) Maitake extract (Yukiguni Maitake Co. Ltd. and Tradeworks Group), (4) Echinacea lipophilic, neutral and acidic extracts (Gaia Herbs), (5) Astragalus water, 50% or 95% ethanol extracts (ICM), (6) Turmeric supercritical (SC) or hydro-ethanolic (HE) extracts (New Chapter) or 60% ethanol extract (ICM) and (7) yeast β-glucan (Biotec Pharmacon). Purified saponin extract QS-21 (Antigenics) and semisynthetic saponin GPI-0100 (Advanced BioTherapies) were used as positive control adjuvants. Sera were analyzed by ELISA against synthetic globo H ceramide or GD3 and KLH. Results Consistent significant adjuvant activity was observed after s.c. vaccination with the Coriolus extracts (especially PSK), a 95% ethanol extract of Astragalus and yeast β-glucan, and (to a lesser extent) Maitake. Antibodies against KLH in all cases and against globo H in most cases were induced by these botanicals. Little or no adjuvant activity was demonstrated with H-48 or Echinacea extracts or the Astragalus water extract. Experiments with GD3–KLH as immunogen confirmed the adjuvant activity of the Coriolus, yeast β-glucan and Astragalus extracts. While extraction with ethanol concentrated the active ingredients in Astragalus, it had no impact on Coriolus where the 90% ethanol precipitate and solute were equally active. Conclusions Some, but not all, botanicals purported to be immune stimulants had adjuvant activity in our model. PSK and Astragalus were surprisingly active and are being further fractionated to identify the most active adjuvant components.

By activating complement, antitumor monoclonal antibodies coat tumor cells with iC3b. beta-glucans, naturally occurring glucose polymers, bind to the lectin domain of the leukocyte receptor CR3, prime it for binding to iC3b, and trigger cytotoxicity of iC3b-coated tumor cells. We studied the combination of the complement-activating antibody rituximab with barley-derived (1-->3),(1-->4)-beta-D-glucan (BG) against CD-20 positive lymphoma xenografts in SCID mice. Growth of established subcutaneous non-Hodgkin's lymphoma (NHL) (Daudi and EBV-derived B-NHL) or Hodgkin's disease (Hs445 and RPMI6666) was significantly suppressed in mice treated with a combination of intravenous rituximab and oral BG, when compared to mice treated with rituximab or BG alone. Survival of mice with disseminated lymphoma was significantly increased in the combination group as compared to other treatment groups. No clinical toxicity was observed. The therapeutic efficacy and lack of toxicity of this combination supports further investigation into its clinical utility.

Gross total resection of the primary tumor in treatment of high-risk neuroblastoma remains controversial. Furthermore, there are few reports of the effect of primary tumor resection on local control as opposed to overall survival. The authors reviewed their institutional experience to assess the effect of primary tumor resection on local control and overall survival.A total of 141 patients were treated on protocol between November 1, 1979 and June 25, 2002 and are the subject of this report. Gross total resection was assessed by review of operative notes, postoperative computerized axial tomograms, and postoperative meta-iodobenzyl guanidine (MIBG)1 scans when available.The median age was 3.3 years, and all patients were International Neuroblastoma Staging System (INSS) stage 4 with 79% having metastases to cortical bone. The primary site was the adrenal gland in 74%, the central abdominal compartment in 13%, the posterior mediastinum in 7%, and other sites in 6%. Gross total resection was accomplished in 103 (73%) but was more than 90% for the last 3 protocols. Five kidneys were lost overall. The probability of local progression was 50% in unresected patients compared with 10% in patients undergoing gross total resection (P.01). Overall survival rate in resected patients was 50% compared with 11% in unresected patients (P.01).Our data indicate that local control and overall survival rate are correlated with gross total resection of the primary tumor in high-risk neuroblastoma. Gross total resection should be part of the management of stage 4 neuroblastoma in patients greater than 1 year of age.

The striking differences in the natural history of clinical subgroups of neuroblastoma (NB), and the evolving therapeutic approaches for each, makes it imperative for prognostic markers to be reevaluated within individual clinical categories. At least one third of NB cases present without distant metastasis and cytotoxic therapy does not alter the natural history. We carried out a retrospective analysis of archived tumor samples. Fifty-seven of these patients had local-regional (LR) NB and were managed conservatively, initially treated with surgery alone. Among the biologic and clinical features analyzed including age, stage, histology, ploidy, MYCN , and 1p36, 1p22, 11q, 14q, 9p and 19q loss of heterozygosity (LOH) in multivariate analysis, diploidy was one of the most significant factors associated with progression-free survival and stage 4 progression. Clonal ploidy heterogeneity was common in LR NB. A predominant near-triploid clonal population was found in most cases of non-progressing LR NB tumors whereas progressing LR NB cases had a predominant diploid clone. We also reviewed the prognostic factors among 84 stage 4 NB cases treated with the N5, N6 or N7 protocols at MSKCC from 1987 to 1999. Traditional markers such as lactate dehydrogenase (LDH), ferritin, age and MYCN status were not prognostic in the univariate analysis. 11q23 and 1p22 LOH were correlated with better survival. These results highlight the evolving significance of prognostic analysis in homogeneous clinical groups undergoing similar treatments. To further characterize the gene expression profile between local-regional and metastatic NB, we carried out Microarray analysis of 41 NB tumors and 12 NB cell lines, using the Affymetrix Genechip Human Genome U95 Set ™ . Distinct gene expression patterns between metastatic and non-metastatic NB tumors have been identified. Validation of these results and further mechanistic studies may shed new light on the biology of metastasis in human NB.

Metastatic solid tumors in children are difficult to cure. Despite intensive chemoradiotherapy plus aggressive surgery, eradicating these diseases has remained a formidable challenge. Differentiation therapy and monoclonal antibody (mAb) may improve the outlook of patients with stage 4 neuroblastoma. The idiotype network appears to represent one component of a complex mechanism for success with mAb immunotherapy. Ongoing strategies to genetically re-engineer mAbs may offer new treatment possibilities against some of these deadly cancers in pediatrics.

Purpose: To evaluate the efficacy of 21 Gy hyperfractionated radiotherapy for local control in conjunction with surgery and intensive systemic therapy for patients with Stage 4 neuroblastoma. Methods and Materials: After achieving a partial or complete remission, 47 children, ages 1–10 years, with Stage 4 neuroblastoma were treated on four consecutive institutional protocols (N4–N7) with dose-intensive multi-agent chemotherapy, maximal surgical debulking, and hyperfractionated radiotherapy (1.5 Gy twice a day to 21 Gy). Radiotherapy fields encompassed the initial tumor volume and regional lymph nodes plus a 3-cm margin. This was followed by consolidation with either autologous bone marrow transplantation (N4 and N5) or immunotherapy (N6 and N7). Results: Forty-five of 47 patients had a complete response to surgery and chemotherapy prior to radiotherapy. Five-year actuarial rates of local control, progression-free survival, and overall survival were 84%, 40%, and 45%, respectively. Among 26 patients who relapsed, 1 failed only at the primary site, 22 developed distant metastases exclusively, and 3 had both local and distant failures. There were no acute complications of radiotherapy. Conclusion: Hyperfractionated radiotherapy to 21 Gy, in conjunction with dose-intensive systemic therapy and aggressive surgical resection, is well tolerated and is associated with durable local control for most patients with Stage 4 neuroblastoma.

Human neuroblastoma cell lines typically consist of heterogenous subpopulations of cells that are morphologically and biochemically distinct. The cell types are characterized as neuroblastic (N-type), substrate-adherent Schwann-like (S-type), or intermediate (I). These cell types can undergo spontaneous or induced transdifferentiation in vitro. We investigated the complement sensitivity of different neuroblastoma cell lines and of matched sets of cloned N- and S-type neuroblastoma cell lines. Human neuroblastoma cell lines that consisted predominantly of a neuroblastic phenotype were shown to be significantly more susceptible to human complement-mediated lysis than cell lines of other cancer types. Complement sensitivity of neuroblastoma cell lines was correlated with low levels of CD59, decay-accelerating factor, and membrane cofactor protein expression. We found that cloned S-type neuroblastoma cells were much more resistant to complement-mediated lysis than cloned N-type cells. The increased complement resistance of S-type cells was shown to be due to increased expression of membrane-bound complement inhibitors. CD59 was the single most important protein in providing S-type cells with protection from complement lysis. S-type cells were also found to express lower levels of GD2, a target antigen for a complement activating monoclonal antibody currently in clinical trials for neuroblastoma immunotherapy. The ability of S-type cells to evade complement, and the ability of S-type cells to differentiate into the more tumorigenic N-type cells, may represent a mechanism of tumor survival and regrowth, a phenomenon often observed with this cancer.

Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1 ), and the non-cytotoxic F(ab′) 2 fragment of the cytotoxic monoclonal anti-G D2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab′) 2 -CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insuffiecient and heterogenous antigen expression on tumor cells for immunotherapy.

The importance of gross total resection of the primary tumor in stage 4 neuroblastoma is controversial. The purpose of this study was to assess the impact of gross total resection of the primary tumor on clinical outcome in patients with stage 4 neuroblastoma diagnosed at more than 1 year of age. The authors retrospectively analyzed 70 newly diagnosed cases treated with one of five regimens of increasing dose-intensity. Outcome variables included survival time from diagnosis, and time to local recurrence or local tumor progression. Patient variables analyzed for impact on survival included age, anatomic location of the primary tumor, radiation treatment of the primary site, complete resection at diagnosis, gross total resection (GTR) at any time in the course of therapy, and treatment protocol dose-intensity. GTR was accomplished in seven patients at the time of diagnosis and in 32 patients after chemotherapy. The likelihood of complete gross resection after chemotherapy increased with greater protocol intensity. The only patient variables that correlated with improved survival were GTR (P = .03) and chemotherapy protocol (P = .01). GTR was also associated with improved local control. Although an independent effect of GTR on survival was not demonstrable because complete resection after chemotherapy correlated strongly with increasing protocol intensity, its association with improved overall survival was striking. These results support a continued role for GTR in high-risk neuroblastoma, along with intensive chemotherapy.