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The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALKF1174L (exon 23 position 3522, C>A) and ALKR1275Q (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status.

PURPOSE Minimal residual disease (MRD) helps to accurately assess when children with late bone marrow relapses of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) will benefit from allogeneic hematopoietic stem-cell transplantation (allo-HSCT). More detailed dissection of MRD response heterogeneity and the specific genetic aberrations could improve current practice. PATIENTS AND METHODS MRD was assessed after induction treatment and at different times during relapse treatment until allo-HSCT (indicated in poor responders to induction; MRD ≥ 10−3) for patients being treated for late BCP-ALL bone marrow relapses (n = 413; median follow-up, 9.4 years) in the ALL-REZ BFM 2002 trial/registry (ClinicalTrials.gov identifier: NCT00114348 ). RESULTS Patients with both good (MRD < 10−3) and poor responses to induction treatment reached excellent event-free survival (EFS; 72% v 65%) and overall survival (OS; 82% v 74%). Patients with MRD of 10−2 or greater after induction had reduced EFS (56%), and their MRD persisted until allo-HSCT more frequently than it did in patients with MRD of 10−3 or greater to less than 10−2 ( P = .037). Patients with 25% or more leukemic blasts after induction (early nonresponders) had the poorest prognosis (EFS, 22%). Interestingly, patients with MRD of 10−3 or greater before allo-HSCT (late nonresponders) still had an EFS of 50% and OS of 63%, which in principle justifies allo-HSCT in these patients. From a panel of selected candidate genes, TP53 alterations (frequency, 8%) were the only genetic alteration with independent prognostic value in any MRD-based response subgroup. CONCLUSION After induction treatment, MRD-based treatment stratification resulted in excellent survival in patients with late relapsed BCP-ALL. Prognosis could be further improved in very poor responders by intensifying treatment directly after induction. TP53 alterations can be defined as a novel genetic high-risk marker in all MRD response groups in late relapsed BCP-ALL. Here we identified early and late nonresponders to be considered as events in future trials.

Simple Summary The childhood tumor, neuroblastoma, has a broad clinical presentation. Risk assessment at diagnosis is particularly difficult in molecularly heterogeneous high-risk cases. Here we investigate the potential of imaging mass spectrometry to directly detect intratumor heterogeneity on the protein level in tissue sections. We show that this approach can produce discriminatory peptide signatures separating high- from low- and intermediate-risk tumors, identify 8 proteins aassociated with these signatures and validate two marker proteins using tissue immunostaining that have promise for further basic and translational research in neuroblastoma. We provide proof-of-concept that mass spectrometry-based technology could assist early risk assessment in neuroblastoma and provide insights into peptide signature-based detection of intratumor heterogeneity. Abstract Risk classification plays a crucial role in clinical management and therapy decisions in children with neuroblastoma. Risk assessment is currently based on patient criteria and molecular factors in single tumor biopsies at diagnosis. Growing evidence of extensive neuroblastoma intratumor heterogeneity drives the need for novel diagnostics to assess molecular profiles more comprehensively in spatial resolution to better predict risk for tumor progression and therapy resistance. We present a pilot study investigating the feasibility and potential of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to identify spatial peptide heterogeneity in neuroblastoma tissues of divergent current risk classification: high versus low/intermediate risk. Univariate (receiver operating characteristic analysis) and multivariate (segmentation, principal component analysis) statistical strategies identified spatially discriminative risk-associated MALDI-based peptide signatures. The AHNAK nucleoprotein and collapsin response mediator protein 1 (CRMP1) were identified as proteins associated with these peptide signatures, and their differential expression in the neuroblastomas of divergent risk was immunohistochemically validated. This proof-of-concept study demonstrates that MALDI-MSI combined with univariate and multivariate analysis strategies can identify spatially discriminative risk-associated peptide signatures in neuroblastoma tissues. These results suggest a promising new analytical strategy improving risk classification and providing new biological insights into neuroblastoma intratumor heterogeneity.

The clinical course of neuroblastoma is more heterogeneous than any other malignant disease. Most low-risk patients experience regression after limited or even no chemotherapy. However, more than half of high-risk patients die from disease despite intensive multimodal treatment. Precise patient characterization at diagnosis is key for risk-adapted treatment. The guidelines presented here incorporate results from national and international clinical trials to produce recommendations for diagnosing and treating neuroblastoma patients in German hospitals outside of clinical trials.

Background Survivors of heritable retinoblastoma carry a high risk to develop second cancers. Eye-preserving radiotherapy raises this risk, while the impact of chemotherapy remains less defined. Procedure This population-based study characterizes the impact of all treatment modalities on second cancers incidence and type after retinoblastoma treatment in Germany. Data on second cancer incidence in 648 patients with heritable retinoblastoma treated between 1940 and 2008 at the German national reference center for retinoblastoma were analyzed to identify associations with treatment. Results The cumulative incidence ratio (per 1,000 person years) of second cancers was 8.6 (95% confidence interval 7.0–10.4). Second cancer incidence was influenced by type of retinoblastoma treatment but not by the year of diagnosis or by sex. Radiotherapy and systemic chemotherapy increased the incidence of second cancers (by 3.0- and 1.8-fold, respectively). While radiotherapy was specifically associated with second cancers arising within the periorbital region in the previously irradiated field, chemotherapy was the strongest risk factor for second cancers in other localizations. Soft tissue sarcomas and osteosarcomas were the most prevalent second cancers (standardized incidence ratio 179.35 compared to the German population). Conclusions Second cancers remain a major concern in heritable retinoblastoma survivors. Consistent with previous reports, radiotherapy increased second cancer incidence and influenced type and localization. However, chemotherapy was the strongest risk factor for second malignancies outside the periorbital region. Our results provide screening priorities during life-long oncological follow-up based on the curative therapy the patient has received and emphasize the need for less-detrimental therapies for children with heritable retinoblastoma.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that is often lethal when relapse occurs. We here used whole-exome sequencing, mRNA expression profiling, array CGH and DNA methylation analysis to characterize 16 paired samples at diagnosis and relapse from individuals with neuroblastoma. The mutational burden significantly increased in relapsing tumors, accompanied by altered mutational signatures and reduced subclonal heterogeneity. Global allele frequencies at relapse indicated clonal mutation selection during disease progression. Promoter methylation patterns were consistent over disease course and were patient specific. Recurrent alterations at relapse included mutations in the putative CHD5 neuroblastoma tumor suppressor, chromosome 9p losses, DOCK8 mutations, inactivating mutations in PTPN14 and a relapse-specific activity pattern for the PTPN14 target YAP. Recurrent new mutations in HRAS, KRAS and genes mediating cell-cell interaction in 13 of 16 relapse tumors indicate disturbances in signaling pathways mediating mesenchymal transition. Our data shed light on genetic alteration frequency, identity and evolution in neuroblastoma.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase that promotes G2/M-phase transition, is expressed in elevated levels in high-risk neuroblastomas and correlates with unfavorable patient outcome. Recently, we and others have presented PLK1 as a potential drug target for neuroblastoma, and reported that the BI2536 PLK1 inhibitor showed antitumoral actvity in preclinical neuroblastoma models. Here we analyzed the effects of GSK461364, a competitive inhibitor for ATP binding to PLK1, on typical tumorigenic properties of preclinical in vitro and in vivo neuroblastoma models. GSK461364 treatment of neuroblastoma cell lines reduced cell viability and proliferative capacity, caused cell cycle arrest and massively induced apoptosis. These phenotypic consequences were induced by treatment in the low-dose nanomolar range, and were independent of MYCN copy number status. GSK461364 treatment strongly delayed established xenograft tumor growth in nude mice, and significantly increased survival time in the treatment group. These preclinical findings indicate PLK1 inhibitors may be effective for patients with high-risk or relapsed neuroblastomas with upregulated PLK1 and might be considered for entry into early phase clinical trials in pediatric patients. OA gold

OA gold The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

The invasive nature of surgical biopsies most often prevents their sequential application to monitor disease in patients with high-risk neuroblastoma. Even when available, single biopsies often fail to reflect neuroblastoma dynamics, intratumor heterogeneity and drug sensitivities likely to change during neuroblastoma evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA (cfDNA) isolated from blood plasma would improve outcome prediction, patient monitoring and treatment selection for high-risk neuroblastoma patients, by providing a method to follow clonal evolution in tumor subpopulations and treatment response as well as capture the molecular landscape of all tumor clones. As a first step, we established a digital droplet PCR (ddPCR) protocol to analyze the MYCN copy number status. Analyzing the dilution series of a synthetic MYCN template consisting of 70 nucleotides demonstrated a strong correlation between theoretically calculated MYCN copy number and the MYCN copy number measured using our ddPCR protocol. We compared MYCN copy number status in a large panel of neuroblastoma cell lines with MYCN copy number determined from cfDNAs isolated from cell culture media from the corresponding cell lines. Our ddPCR protocol reliably detected MYCN status in the cell line using cfDNA analysis. Next, we turned to a subcutaneous MYCN-driven neuroblastoma xenograft model to compare MYCN copy number in the xenografted tumor to MYCN copy number determined from cfDNA isolated from the mouse blood plasma. MYCN copy numbers also positively correlated using this model. We then analyzed MYCN copy number status in primary neuroblastoma samples from 10 patients and compared these values to the MYCN copy number determined from cfDNA isolated from 200 µl of blood plasma collected from the corresponding patients. These final validation steps demonstrated that our ddPCR protocol for cfDNA from patient blood plasma reliably detects MYCN copy number status in the tumor. Our data justify the further development of molecular neuroblastoma characterization from cfDNA in patient blood plasma. An expanded molecular diagnostic monitoring palette will improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients. Citation Format: Marco Lodrini, Annika Sprüssel, Kathy Astrahantseff, Daniela Tiburtius, Robert Konschak, Holger Lode, Matthias Fischer, Johannes H. Schulte, Angelika Eggert, Hedwig Deubzer. Using droplet digital PCR to analyze MYCN copy number in plasma from patients with high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5679. doi:10.1158/1538-7445.AM2017-5679

The gene expressing the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) is mutated and aberrantly expressed in several cancers. The clinical efficacy of the ALK inhibitor, crizotinib, lags behind expectations for treating MYCN-amplified, ALK-mutant neuroblastoma, a deadly childhood cancer. In this issue of Science Signaling, Umapathy et al. identify the kinase extracellular signal-regulated kinase 5 (ERK5) as a central mediator that enables ALK to boost MYCN expression, and they show that inhibiting ERK5 in concert with ALK reduced neuroblastoma cell viability in vitro and in xenograft tumor models. This report has important clinical implications for the treatment of patients with neuroblastoma or other tumors that overexpress MYC(N) and harbor ALK mutations, such as non-small-cell lung cancer.

The prognosis for children with high-risk relapsed acute lymphoblastic leukemia (ALL) is poor. Here, we assessed the prognostic importance of response during induction and consolidation treatment prior to hematopoietic stem cell transplantation (HSCT) aiming to evaluate the best time to assess minimal residual disease (MRD) for intervention strategies and in future trials in high-risk ALL relapse patients. Included patients (n=125) were treated uniformly according to the ALL-REZ BFM (Berlin-Frankfurt-Munster) 2002 relapse trial (median follow-up time=4.8 years). Patients with MRD ⩾10(-3) after induction treatment (76/119, 64%) or immediately preceding HSCT (19/71, 27%) had a significantly worse probability of disease-free survival 10 years after relapse treatment begin, with 26% (±6%) or 23% (±7%), respectively, compared with 58% (±8%) or 48% (±7%) for patients with MRD

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ?-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies. OA hybrid

✓ Alterations in the epidermal growth factor receptor (EGFR) and its main ligand, transforming growth factor-α (TGFα), were investigated for a possible prognostic relevance in 125 astrocytic gliomas (44 World Health Organization (WHO) Grade II, 19 WHO Grade III, and 62 WHO Grade IV tumors). The TGFα and EGFR proteins were detected immunohistochemically using monoclonal antibodies. A positive immunoreaction to TGFa was detected in 33 (75%) of 44 WHO Grade II astrocytomas, 18 (95%) of 19 WHO Grade III astrocytoma, and 50 (81%) of 62 WHO Grade IV glioblastomas. No correlation between TGFα immunoreaction and duration of survival could be found. A positive EGFR immunoreaction was detected in seven (16%) of 44 WHO Grade II astrocytomas, five (26%) of 19 WHO Grade III astrocytomas, and 32 (52%) of 62 WHO Grade IV glioblastomas. Of these gliomas, 97 (26 WHO Grade II, 17 WHO Grade III, and 54 WHO Grade IV gliomas) were examined for EGFR gene amplification using a differential polymerase chain reaction assay. Amplification of the EGFR gene was detected in none of the WHO Grade II astrocytomas, one (6%) of 17 WHO Grade III astrocytomas, and 18 (33%) of 54 WHO Grade IV glioblastomas. Twenty-two of the tumors investigated showed a positive EGFR immunoreaction without detectable gene amplification (five WHO Grade II, four WHO Grade III, and 13 WHO Grade IV tumors). Gene amplification was invariably associated with a positive EGFR immunoreaction. For the entire study group, a strong correlation between EGFR alterations (gene amplification and positive immunoreaction) and survival could be found. However, this correlation only reflected the higher percentages of cases with EGFR alterations in malignant gliomas and was not an independent prognostic factor as determined by multifactorial analysis. These data demonstrate that EGFR alterations are frequent events in astrocytic gliomas and are largely restricted to glioblastomas. However, within one tumor grade they do not provide prognostic information.

Treatment for neuroblastoma, the most common extracranial childhood tumor, spans a broad range of aggressiveness that mirrors the risk profiles of disease subtypes, with high-risk neuroblastoma still presenting a clinical challenge. Currently, most patients with relapsed neuro-blastoma die of disease and present a major challenge for treatment. New therapeutic options are urgently needed to improve patient survival. Activating mutations in the gene encoding the anaplastic lymphoma kinase (ALK) remain the most frequent druggable mutations identified in neuroblastomas to date. Preclinical data support an oncogene addiction of neuroblastoma cells to mutated ALK and demonstrate that ALK inhibitory therapy strongly combats tumor models. Most recently, pediatric phase I testing has been completed for the first approved ALK inhibitor, Crizotinib, showing very encouraging antitumoral results in neuroblastoma patients. Subsequently, an international phase I study with the second generation ALK inhibitor, LDK-378, will be launched that makes ALK inhibitory therapy also available to pediatric patients in Germany.

Purpose Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary. Methods We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC). Results Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression. Conclusion TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.

MYCN amplification is a common feature of aggressive tumour biology in neuroblastoma. The MYCN transcription factor has been demonstrated to induce or repress expression of numerous genes. MicroRNAs (miRNA) are a recently discovered class of short RNAs that repress translation and promote mRNA degradation by sequence-specific interaction with mRNA. Here, we sought to analyse the role of MYCN in regulation of miRNA expression. Using a miRNA microarray containing 384 different miRNAs and a set of 160 miRNA real-time PCR assays to validate the microarray results, 7 miRNAs were identified that are induced by MYCN in vitro and are upregulated in primary neuroblastomas with MYCN amplification. Three of the seven miRNAs belong to the miR-106a and miR-17 clusters, which have previously been shown to be regulated by c-Myc. The miR-17–92 polycistron also acts as an oncogene in haematopoietic progenitor cells. We show here that miR-221 is also induced by MYCN in neuroblastoma. Previous studies have reported miR-221 to be overexpressed in several other cancer entities, but its regulation has never before been associated with Myc. We present evidence of miRNA dysregulation in neuroblastoma. Additionally, we report miRNA induction to be a new mechanism of gene expression downregulation by MYCN. © 2007 Wiley-Liss, Inc.

MYCN transcriptionally represses CD9 to trigger an invasion-metastasis cascade in neuroblastoma Johannes Fabian, Desiree Opitz, Kristina Althoff, Marco Lodrini, Kathy Astrahantseff, Barbara Hero, Ruth Volland, Anneleen Beckers, Katleen de Preter, Nitin S Patil, Mohammed L Abba, Theresa M Thole, Jasmin Wunschel, Annette Kunkele, Jamie Hu, Leonille Schweizer, Gunhild Mechtersheimer, Daniel R Carter, Belamy B Cheung, Odilia Popanda, Andreas von Deimling, Kai-Oliver Henrich, Frank Westermann, Manfred Schwab, Jan Koster, Rogier Versteeg, Glenn M Marshall, Frank Speleman, Margot Zoeller, Heike Allgayer, Matthias Fischer, Frank Berthold, Andreas E Kulozik, Olaf Witt, Angelika Eggert, Johannes H Schulte, Hedwig E Deubzer