MYCN Gain Research Articles

18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) imaging of pediatric neuroblastoma: a multi-omics parameters method to predict MYCN copy number category.

The MYCN copy number category is closely related to the prognosis of neuroblastoma (NB). Therefore, this study aimed to assess the predictive ability of 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) radiomic features for MYCN copy number in NB. A retrospective analysis was performed on 104 pediatric patients with NB that had been confirmed by pathology. To develop the Bio-omics model (B-model), which incorporated clinical and biological aspects, PET/CT radiographic features, PET quantitative parameters, and significant features with multivariable stepwise logistic regression were preserved. Important radiomics features were identified through least absolute shrinkage and selection operator (LASSO) and univariable analysis. On the basis of radiomics features obtained from PET and CT scans, the radiomics model (R-model) was developed. The significant bio-omics and radiomics features were combined to establish a Multi-omics model (M-model). The above 3 models were established to differentiate MYCN wild from MYCN gain and MYCN amplification (MNA). The calibration curve and receiver operating characteristic (ROC) curve analyses were performed to verify the prediction performance. Post hoc analysis was conducted to compare whether the constructed M-model can distinguish MYCN gain from MNA. The M-model showed excellent predictive performance in differentiating MYCN wild from MYCN gain and MNA, which was better than that of the B-model and R-model [area under the curve (AUC) 0.83, 95% confidence interval (CI): 0.74-0.92 vs. 0.81, 95% CI: 0.72-0.90 and 0.79, 95% CI: 0.69-0.89]. The calibration curve showed that the M-model had the highest reliability. Post hoc analysis revealed the great potential of the M-model in differentiating MYCN gain from MNA (AUC 0.95, 95% CI: 0.89-1). The M-model model based on bio-omics and radiomics features is an effective tool to distinguish MYCN copy number category in pediatric patients with NB.

Aqueous Humor Liquid Biopsy as a Companion Diagnostic for Retinoblastoma: Implications for Diagnosis, Prognosis, and Therapeutic Options: Five Years of Progress

To define the prospective use of the aqueous humor (AH) as a molecular diagnostic and prognostic liquid biopsy for retinoblastoma (RB). This is a prospective, observational study wherein an AH liquid biopsy is performed at diagnosis and longitudinally through therapy for patients with RB. Tumor-derived cell-free DNA is isolated and sequenced for single nucleotide variant analysis of the RB1 gene and detection of somatic copy number alterations (SCNAs). The SCNAs are used to determine tumor fraction (TFx). Specific SCNAs, including 6p gain and focal MycN gain, along with TFx, are prospectively correlated with intraocular tumor relapse, response to therapy, and globe salvage. A total of 26 eyes of 21 patients were included with AH taken at diagnosis. Successful ocular salvage was achieved in 19 of 26 (73.1%) eyes. Mutational analysis of 26 AH samples identified 23 pathogenic RB1 variants and 2 focal RB1 deletions; variant allele fraction ranged from 30.5% to 100% (median 93.2%). At diagnosis, SCNAs were detectable in 17 of 26 (65.4%) AH samples. Eyes with 6p gain and/or focal MycN gain had significantly greater odds of poor therapeutic outcomes (odds ratio = 6.75, 95% CI = 1.06-42.84, P = .04). Higher AH TFx was observed in eyes with vitreal progression (TFx = 46.0% ± 40.4) than regression (22.0 ± 29.1; difference: -24.0; P = .049). Establishing an AH liquid biopsy for RB is aimed at addressing (1) our inability to biopsy tumor tissue and (2) the lack of molecular biomarkers for intraocular prognosis. Current management decisions for RB are made based solely on clinical features without objective molecular testing. This prognostic study shows great promise for using AH as a companion diagnostic. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Simultaneous Copy Number Alteration and Single-Nucleotide Variation Analysis in Matched Aqueous Humor and Tumor Samples in Children with Retinoblastoma.

Retinoblastoma (RB) is a childhood cancer that forms in the developing retina of young children; this tumor cannot be biopsied due to the risk of provoking extraocular tumor spread, which dramatically alters the treatment and survival of the patient. Recently, aqueous humor (AH), the clear fluid in the anterior chamber of the eye, has been developed as an organ-specific liquid biopsy for investigation of in vivo tumor-derived information found in the cell-free DNA (cfDNA) of the biofluid. However, identifying somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) of the RB1 gene, typically requires either: (1) two distinct experimental protocols-low-pass whole genome sequencing for SCNAs and targeted sequencing for SNVs-or (2) expensive deep whole genome or exome sequencing. To save time and cost, we applied a one-step targeted sequencing method to identify both SCNAs and RB1 SNVs in children with RB. High concordance (median = 96.2%) was observed in comparing SCNA calls derived from targeted sequencing to the traditional low-pass whole genome sequencing method. We further applied this method to investigate the degree of concordance of genomic alterations between paired tumor and AH samples from 11 RB eyes. We found 11/11 AH samples (100%) had SCNAs, and 10 of them (90.1%) with recurrent RB-SCNAs, while only nine out of 11 tumor samples (81.8%) had positive RB-SCNA signatures in both low-pass and targeted methods. Eight out of the nine (88.9%) detected SNVs were shared between AH and tumor samples. Ultimately, 11/11 cases have somatic alterations identified, including nine RB1 SNVs and 10 recurrent RB-SCNAs with four focal RB1 deletions and one MYCN gain. The results presented show the feasibility of utilizing one sequencing approach to obtain SCNA and targeted SNV data to capture a broad genomic scope of RB disease, which may ultimately expedite clinical intervention and be less expensive than other methods.

The Gain of the Short Arm of Chromosome 2 (2pgain) Plays an Important Role in CLL Drug Resistance, and Has to be Systematically Considered before Deciding an Adapted Therapy

The Gain of the Short Arm of Chromosome 2 (2pgain) Plays an Important Role in CLL Drug Resistance, and Has to be Systematically Considered before Deciding an Adapted Therapy

Selected Articles from This Issue

BRAF mutation is a common driver event in melanoma, rendering the tumors reliant on constitutive signaling through the MAp. pathway and the extracellular signal-regulated kinases (ERK) 1 and 2. Thus, MAPK pathway inhibitors that act through both ERK1 and ERK2 have become a key component of the therapeutic arsenal against melanoma, but their use is associated with dose-limiting toxicities. Here, Crowe and colleagues demonstrate that BRAF-mutant melanoma is specifically reliant on ERK2 signaling. Targeted ERK2 ablation resulted in transcriptomic and proteomic alterations that aligned with those effected by full MAPK pathway inhibitors, whereas targeted ERK1 ablation produced a distinct set of responses. Both isoforms promoted cell viability, but ERK2 loss suppressed MAPK pathway activity and cell proliferation more effectively than ERK1 loss. Moreover, ERK2 was shown to compensate for ERK1 depletion in the context of BRAF mutation, whereas ERK1 was not able to compensate for loss of ERK2 activity. The authors argue that these findings support the development of ERK2-selective inhibitors, which could potentially be achieved through the targeting of protein-protein interaction domains that are differentially utilized between the two isoforms.Mutation of DNA damage-responsive Fanconi Anemia family members (FANC) is associated with hereditary cancer syndromes and familial cancers, particularly breast and ovarian cancer, due to their role in mediating homologous recombination repair. In particular, FANCJ has been nominated as a key tumor suppressor in several cancer types, though functional data on the many clinically identified FANCJ mutations are lacking. Here, Calvo and colleagues employ a mutation library to screen for loss-of-function (LOF) mutations that confer sensitivity to interstrand crosslinking (ICL) agents. They find that missense and nonsense mutations in the helicase domain are associated with FANCJ LOF, whereas mutations outside this domain did not affect the response to ICL chemotherapeutics. Notably, mutations in the carboxy-terminal domain, which mediates protein-protein interactions with other homologous recombination mediators such as BRCA1, did not significantly affect FANCJ function in response to ICL agents. Finally, while only a minority of FANCJ mutations (∼12%) exhibited LOF in response to ICL, the authors note that the comprehensive mutation library developed here will continue to be useful in characterizing FANCJ mutants and their functionality in response to other stimuli and other types of genomic lesions.Circulating tumor cells (CTC) provide important prognostic information in metastatic castration-resistant prostate cancer (mCRPC), in which the expression of androgen receptor (AR) splice variants is a key prognostic indicator of patient response to enzalutamide and/or abiraterone (enza/abi). However, most patients do not express AR variants, and therefore additional markers are needed. In this study, Gupta et al. leverage CTC exome sequencing and cop. number alteration profiles from men with mCRPC undergoing therapy with enza/abi to identify genomic events associated with therapy resistance. The authors identify a subset of patients who progressed rapidly on enza/abi and bore genomic lesions that were detectable in pooled CTC DNA, including gain of MYCN, BRCA2, and AR, and loss of CHD1, NCOR2, and PTEN. In patients who initially responded and later acquired resistance, CTC sampling uncovered mutations and copy number alterations which converged on genes linked to proliferation, hormone signaling, lineage plasticity and epigenetic signaling, and resistance to cell death. Taken together, these data could form the molecular basis for more effective therapeutic decision making in mCRPC.Tumors commonly down-regulate antigen processing and presentation pathways in order to evade the anti-tumor immune response, which can undermine the efficacy of immune checkpoint blockade. Here, Zhang and colleagues show that Qa-1b—the mouse homolog of the human major histocompatibility complex class I molecule HLA-E—is a key player in how tumors and the immune system interact when antigen presentation is defective. Loss of TAP1 resulted in the establishment of an immunosuppressive tumor microenvironment, but loss of both TAP1 and Qa-1b restored sensitivity to immunotherapy. This effect was mediated at least in part through direct interactions between Qa-1b and natural killer cells, likely through the inhibitory NKG2A receptor through which Qa-1b and HLA-E normally act. Concurrent disruption of TAP1 and Qa-1b reversed immunoresistance by causing increased CD8+ T cell to Treg ratios, increased natural killer cell infiltration and activity, and sensitization to immune checkpoint blockade with anti-PD-1 antibodies. The data suggest that Qa-1b and HLA-E engage in crosstalk with the antigen presentation machinery and represent a key control node that governs the immune response to antigen presentation by tumor cells.

Abstract 186: Gain of chromosome 6p is a molecular biomarker for prognostication of retinoblastoma ocular survival: The aqueous humor surrogate tumor biopsy

Abstract Purpose: Aqueous humor (AH) is a novel source of tumor-derived cell-free DNA (cfDNA) and can serve as a surrogate tumor biopsy, or liquid biopsy, for retinoblastoma (RB). We previously identified a somatic copy number alteration (SCNA) gain of chromosome 6p as associated with a 10-fold increased risk of enucleation. In this study, we provide a 2-year extended update to further explore 6p gain as a prognostic biomarker for ocular survival. Methods: Patients diagnosed with RB from 12/2014 - 7/2019 from whom AH was sampled were included. AH was extracted via clear corneal paracentesis at diagnosis, during intravitreal injection of chemotherapy or enucleation. CfDNA was extracted; shallow whole genome sequencing was performed to assess for cfDNA tumor fractions and highly recurrent RB SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). Patient demographics, treatment regimen and globe salvage were recorded. Results: 50 eyes from 46 patients were included: 27 eyes were salvaged and 23 were enucleated. Most eyes (36/50,72%) were TNM stage cT2b. 116 samples of AH were analyzed. There were no cases of orbital relapse or metastatic spread. The median follow-up was 30 months (range 6-64 mos). After genomic evaluation, 33 / 50 eyes (66%) demonstrated one or more highly-recurrent RB SCNAs and 39/50 (78%) demonstrated any measurable SCNA. Gain of 6p was the most prevalent RB SCNA among all 50 eyes (50%), followed by 1q gain (38%), 16q loss (30%), 2p gain (including 3 focal MYCN gains; 18%), and 13q loss (16%). RB SCNAs were significantly more prevalent in enucleated eyes (21/23, 91.3%) than in salvaged eyes (12/27, 44.4%; Fisher's exact, P=0.0007). Of these, 6p gain was particularly more prevalent in enucleated eyes (17/23, 73.9%) than in salvaged eyes (8/27, 29.6%; Chi-squared, P=0.002) Of all the RB SCNAs, the presence of 6p gain in the cfDNA in the AH portended nearly 10 times greater odds of being enucleated (OR=9.87,95% CI=1.75-55.65, P=0.009). No single other SCNA had a statistically significant power to predict enucleation although there was a trend towards significance for 2p/focal MYCN gain (P=0.09) limited by the few number of eyes with this SCNA. Conclusions: Although we previously identified chromosomal gain of 6p as a potential indicator of an aggressive phenotype in RB, that study included a smaller cohort of patients and limited follow-up. With extended follow-up and nearly double the number of eyes and AH samples, the data continue to demonstrate that AH is a high-yield surrogate tumor biopsy, and the presence of RB-specific SCNAs–specifically gain of 6p–has the potential to serve as a prognostic biomarker for RB. Citation Format: Jesse L. Berry, Liya Xu, Ashley Polski, Rishvanth Prabakar, Mark Reid, Jonathan Kim, Peeter Kuhn, James Hicks. Gain of chromosome 6p is a molecular biomarker for prognostication of retinoblastoma ocular survival: The aqueous humor surrogate tumor biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 186.

Circulating tumor cell (CTC) genomic signatures of hormone therapy resistance in men with metastatic castration-resistant prostate cancer (mCRPC).

147 Background: Men with CTC AR-V7 + mCRPC have very poor outcomes when treated with enzalutamide/abiraterone. However, many men lack AR-V7. Here, we determined whether baseline or post-treatment DNA alterations in CTCs from AR-V7 negative mCRPC men could provide clinical utility in predicting outcomes with these hormonal therapies. Methods: We analyzed whole-genome copy number alterations (CNA) using array-comparative genomic hybridization (aCGH) in CTCs from 48 men (45 baseline and 28 progression), and whole-exome sequencing (WES) from 11 mCRPC men treated with abi/enza, longitudinally, and focused exclusively on AR-V7 negative men (N = 40) by the Epic-AR-V7 nuclear protein assay, and comparing those men who benefit from therapy vs. who do not. Results: We observed broad heterogeneity of CNAs between patients; common genomic alterations included gain in KDM6A (44%), FOXA1 (44%), MYCN (32%), and AR (38%), and loss in BRCA1 (30%) and PTEN (25%). Men who had the clinical benefit to abi/enza (n = 23, median PFS 10 mo) were more likely to have CTCs with genomic gains of ATM, HSD17B4, or PTEN, and loss of BRAF, ABL1, or NKX3-1. Likewise, men who did not benefit from abi/enza (n = 14, median PFS 2.6 mo) had CTCs with more copy number alterations than men who had clinical benefit (median 19.5 vs. 14, p = 0.01), and were enriched for gain of BRCA2, APC, KDM5D, SPARC, MYCN, AR, and CYP11B1, and loss of PTEN, CHD1, PHLPP1, and NCOR2. After progression on abi/enza, we observed clonal evolution of CTCs harboring gain of ATM, FOXA1, KDM6A, CYP11B1, MYC, APC, and NCOR2, and loss of NCOR1, ERG, and RUNX2. Several COSMIC-validated non-synonymous pathogenic exome mutations were detected in progressed or non-responding patients’ CTC DNA; TP53 (55 vs 27% at baseline), AKAP9 (36 vs 9%), CDK12, KMT2D, and BRAF (each 36 vs 18%), and BRD4 and SPOP (each 18 vs 0%). Conclusions: We demonstrate that specific CTC genomic profiles associated with TP53, PTEN, WNT, DNA repair, epigenetic, and AR signaling, as well as lineage plasticity pathways are associated with worse clinical outcomes in AR-V7 negative men with mCRPC treated with abi/enza. Further mechanistic and validation studies are warranted. Clinical trial information: NCT02269982.

Copy Number Variations in Wilms Tumor: A Pilot Study From India.

The prognostic role of copy number variation is upcoming in Wilms tumor, and its identification will help in tailored therapy for improved cure. This was a retrospective, nested case-control, pilot study. Formalin-fixed paraffin-embedded blocks of nephrectomy specimens were retrieved for the study and control groups (children with relapse and survivors for ≥2 y). Multiplex ligand probe amplification (MRC-Holland probe-mix P 380 A1) was performed, with 3 reference samples of normal kidney DNA run for every 7 cases. At least 1 variation was detected in 41 (97.8%) specimens. Loss of heterozygosity 1p was not observed. Loss of 16q, 1q gain, and MYCN gain were observed in 5 (11.9%), 29 (69%), and 39 (92.9%) specimens, respectively. The occurrence of copy number variations was similar in both groups: 1q gain: 15 versus 14 (P=1.0), 16q loss: 4 versus 1 (P=0.34), MYCN gain: 19 versus 20 (P=1.0). The gain of 1q, 16p loss, and MYCN gain did not differ across stage or age. The gain of 1q, MYCN gain, and 16p loss were identified. A higher occurrence of 1q gain and MYCN gain and a lack of difference in the distribution of variations among survivors and those with a relapse suggest a different molecular profile of Wilms tumor in Indian children.

Discordant and heterogeneous clinically relevant genomic alterations in circulating tumor cells vs plasma DNA from men with metastatic castration resistant prostate cancer.

Circulating tumor cell (CTC) and cell-free (cf) DNA-based genomic alterations are increasingly being used for clinical decision-making in oncology. However, the concordance and discordance between paired CTC and cfDNA genomic profiles remain largely unknown. We performed comparative genomic hybridization (CGH) on CTCs and cfDNA, and low-pass whole genome sequencing (lpWGS) on cfDNA to characterize genomic alterations (CNA) and tumor content in two independent prospective studies of 93 men with mCRPC treated with enzalutamide/abiraterone, or radium-223. Comprehensive analysis of 69 patient CTCs and 72 cfDNA samples from 93 men with mCRPC, including 64 paired samples, identified common concordant gains in FOXA1, AR, and MYC, and losses in BRCA1, PTEN, and RB1 between CTCs and cfDNA. Concordant PTEN loss and discordant BRCA2 gain were associated with significantly worse outcomes in Epic AR-V7 negative men with mCRPC treated with abiraterone/enzalutamide. We identified and externally validated CTC-specific genomic alternations that were discordant in paired cfDNA, even in samples with high tumor content. These CTC/cfDNA-discordant regions included key genomic regulators of lineage plasticity, osteomimicry, and cellular differentiation, including MYCN gain in CTCs (31%) that was rarely detected in cfDNA. CTC MYCN gain was associated with poor clinical outcomes in AR-V7 negative men and small cell transformation. In conclusion, we demonstrated concordance of multiple genomic alterations across CTC and cfDNA platforms; however, some genomic alterations displayed substantial discordance between CTC DNA and cfDNA despite the use of identical copy number analysis methods, suggesting tumor heterogeneity and divergent evolution associated with poor clinical outcomes.

Abstract 2523: Targeted exome sequencing profiles genetic alterations in oral squamous cell carcinoma from Japanese patients

Abstract Somatic mutation analysis is a standard practice in the study of human cancers to identify mutations that cause therapeutic sensitization and resistance. We performed comprehensive genomic analyses that used PCR target enrichment and next-generation sequencing on Ion Torrent semiconductor sequencers. Forty-seven oral squamous cell cancer (OSCC) samples and their corresponding noncancerous tissues were used for multiplex PCR amplification to obtain targeted coverage of the entire coding regions of 409 cancer-related genes (covered regions: 95.4% of total, 1.69 megabases of target sequence). The number of somatic mutations in 47 patients with OSCC ranged from 1 to 20 with a mean of 7.60. The most frequent mutations were in TP53 (61.7%), NOTCH1 (25.5%), CDKN2A (19.1%), SYNE1 (14.9%), PIK3CA (10.6%), ROS1 (10.6%), and TAF1L (10.6%). The majority of TP53 mutations (83.9%) are localized in the DNA binding domain of the protein (residues 100-300). Importantly, TP53 mutations tended to be more frequent in HPV-negative OSCCs compared to HPV-positive cases (p = 0.00026). We also detected copy number variations (CNVs) in the segments of the genome that could be duplicated or deleted from deep sequencing data. The genes most frequently affected were EGFR (gain in 38.3%) and CCND1 (gain in 34.0%), followed by PIK3CA (gain in 31.9%), RB1 (loss in 27.7%), ERBB2 (gain in 23.4%), MYC (gain in 23.4%), CCND2 (gain in 17.0%), MYCN (gain in 14.9%), ATM (loss in 14.9%), MDM2 (gain in 14.9%), CDKN2A (loss in 14.9%), NRAS (gain in 12.8%), HRAS (gain in 12.8%), BCL2L1 (gain in 12.8%), and KRAS (gain in 8.5%). We validated 100%, 100%, 83.3%, 83.3% and 100% of the OSCC tissues that showed CNV gain for CCND1, CCND2, HRAS, NRAS and KRAS respectively in quantitative PCR copy number analysis. Pathway assessment showed that the somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation and RTK-MAPK-PI3K. Univariate survival analysis revealed that mutations in NOTCH1 and PIK3CA were found to be associated with worse overall survival in OSCC patients (P = 0.0054 and P = 0.0162, respectively). Interestingly, RTK-MAPK-PI3K pathway alteration correlated to poor survival, suggesting that RTK-MAPK-PI3K pathway is potentially a therapeutic target in this disease. We detected the tumor-specific TP53 mutation in the plasma cell-free DNA from two OSCC patients, and after surgery, the test for these mutations became negative. This study may enable better selection of therapies and deliver improved outcomes for OSCC patients when combined with clinical diagnostics. Citation Format: Yasushi Sasaki, Takafumi Nakagaki, Kazuhiro Ogi, Masashi Idogawa, Takashi Tokino. Targeted exome sequencing profiles genetic alterations in oral squamous cell carcinoma from Japanese patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2523.

Clinical and molecular features of treatment-related neuroendocrine prostate cancer.

Treatment-related neuroendocrine prostate cancer is a lethal form of prostate cancer that emerges in the later stages of castration-resistant prostate cancer treatment. Treatment-related neuroendocrine prostate cancer transdifferentiates from adenocarcinoma as an adaptive response to androgen receptor pathway inhibition. The incidence of treatment-related neuroendocrine prostate cancer has been rising due to the increasing use of potent androgen receptor pathway inhibitors. Typically, treatment-related neuroendocrine prostate cancer is characterized by either low or absent androgen receptor expression, small cell carcinoma morphology and expression of neuroendocrine markers. Clinically, it manifests with predominantly visceral or lytic bone metastases, bulky tumor masses, low prostate-specific antigen levels or a short response duration to androgen deprivation therapy. Furthermore, although the tumor initially responds to platinum-based chemotherapy, the duration of the response is short. Based on the poor prognosis, it is imperative to identify novel molecular targets for treatment-related neuroendocrine prostate cancer. Recent advances in genomic and molecular research, supported by novel invivo models, have identified some of the key molecular characteristics of treatment-related neuroendocrine prostate cancer. The gain of MYCN and AURKA oncogenes, along with the loss of tumor suppressor genes TP53 and RB1 are key genomic alterations associated with treatment-related neuroendocrine prostate cancer. Androgen receptor repressed genes, such as BRN2 and PEG10, are also necessary for treatment-related neuroendocrine prostate cancer. These genetic changes converge on pathways upregulating genes, such as SOX2 and EZH2, that facilitate lineage plasticity and neuroendocrine differentiation. As a result, on potent androgen receptor pathway inhibition, castration-resistant prostate cancer transdifferentiates to treatment-related neuroendocrine prostate cancer in a clonally divergent manner. Further understanding of the disease biology is required to develop novel drugs and biomarkers that would help treat this aggressive prostate cancer variant.

Association of MYCN copy number with clinical features, tumor biology, and outcomes in neuroblastoma: A report from the Children's Oncology Group

High-level MYCN amplification (MNA) is associated with poor outcome and unfavorable clinical and biological features in patients with neuroblastoma. To the authors' knowledge, less is known regarding these associations in patients with low-level MYCN copy number increases. In this retrospective study, the authors classified patients has having tumors with MYCN wild-type tumors, MYCN gain (2-4-fold increase in MYCN signal compared with the reference probe), or MNA (>4-fold increase). Tests of trend were used to investigate ordered associations between MYCN copy number category and features of interest. Log-rank tests and Cox models compared event-free survival and overall survival by subgroup. Among 4672 patients, 3694 (79.1%) had MYCN wild-type tumors, 133 (2.8%) had MYCN gain, and 845 (18.1%) had MNA. For each clinical/biological feature, the percentage of patients with an unfavorable feature was lowest in the MYCN wild-type category, intermediate in the MYCN gain category, and highest in the MNA category (P<.0001), except for 11q aberration, for which the highest rates were in the MYCN gain category. Patients with MYCN gain had inferior event-free survival and overall survival compared with those with MYCN wild-type. Among patients with high-risk disease, MYCN gain was associated with the lowest response rate after chemotherapy. Patients with non-stage 4 disease (according to the International Neuroblastoma Staging System) and patients with non-high-risk disease with MYCN gain had a significantly increased risk for death, a finding confirmed on multivariable testing. Increasing MYCN copy number is associated with an increasingly higher rate of unfavorable clinical/biological features, with 11q aberration being an exception. Patients with MYCN gain appear to have inferior outcomes, especially in otherwise more favorable groups. Cancer 2017;123:4224-4235. © 2017 American Cancer Society.

Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma.

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between MYCN and ALK amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported MYCN and ALK status were detected, including a high-level MYCN amplification in NB-1, a MYCN gain in SH-SY5Y, a high-level ALK amplification in IMR-32 and ALK gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. MYCN and ALK status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. MYCN and ALK copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished MYCN and ALK copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.

Abstract 685: GPC2 is an oncogene and immunotherapeutic target in high-risk neuroblastoma

Abstract Background: GD2-directed immunotherapeutic strategies have improved outcomes in neuroblastoma; however, the majority of patients treated suffer relapse and GD2 expression on pain fibers causes dose-limiting toxicities. Methods: To identify alternative cell surface immunotherapeutic targets, we compared high-risk neuroblastoma (n=126 tumors) and normal tissue RNA sequencing data (GTEx; n=7859 samples from 31 normal tissues) and prioritized genes by differential and absolute expression and cell surface prediction. Genes were further surveyed for somatic copy number gain and correlative expression with MYCN amplification. Differential protein expression and localization were confirmed in neuroblastoma primary tumors (n=98), patient-derived xenografts (n=32; PDXs), cell lines (n=23), and normal pediatric tissues (n=36). Cell lines were subjected to candidate gene gain and loss of function studies (n=11). Additional pediatric tumor RNA sequencing data was surveyed followed by confirmatory immunohistochemistry (IHC). Finally, candidate specific antibodies were isolated from a human Fab phage library and utilized for antibody-drug conjugate (ADC) engineering followed by cytotoxicity studies. Results: We identified 33 differentially expressed cell surface molecules from which we prioritized glypican-2 (GPC2) for validation given GPC2’s robust differential expression (log-fold change tumor vs. normal tissue = 1.71-9.22; p=1.99 x 10-9-1.88 x10-300), high-level absolute RNA expression (median FPKM=60), and frequent DNA copy number gain associated with higher GPC2 expression (35%, n=182 tumors; p&amp;lt;0.005). GPC2 expression was also higher in MYCN amplified neuroblastomas (p&amp;lt;0.05), MYCN binds the GPC2 promoter shown by chromatin immunoprecipitation (ChIP) sequencing and reporter assays, and MYCN depletion resulted in decreased GPC2 expression. Immunoblot, flow cytometry, immunofluorescence, and IHC analysis of primary tumors, PDXs, and cell lines confirmed dense cell surface GPC2 expression. Medulloblastomas (n=62) were also found to have high GPC2 expression that positively correlated with MYC, MYCN, and GPC2 loci gain (p&amp;lt;0.0001). Pediatric normal tissues had very restricted cell surface GPC2 expression, with only low levels found in the esophagus and skin. GPC2 depletion in neuroblastoma cell lines resulted in apoptosis and growth inhibition and GPC2 forced over-expression increased neuroblastoma cell proliferation (p&amp;lt;0.001 for all assays). Finally, a human GPC2 antibody, D3-GPC2-Fab, was developed and shown to bind GPC2 with high affinity and specificity. D3-GPC2-IgG1 induced internalization of GPC2 and was conjugated to pyrrolobenzodiazepine (PBD) dimers to form an ADC which induced potent and specific cytotoxicity to GPC2 expressing neuroblastoma cells (IC50 = 1.7-11 pM). Conclusions: GPC2 is an oncogene and immunotherapeutic target in neuroblastoma and potentially other cancers. Citation Format: Kristopher R. Bosse, Pichai Raman, Maria Lane, Robyn T. Sussman, Jo Lynne Harenza, Daniel Martinez, Sabine Heitzeneder, Zhongyu Zhu, Komal Rathi, Michael Randall, Laura Donovan, Sorana Morrissy, Doncho V. Zhelev, Yang Feng, Jennifer Hwang, Yanping Wang, Bruce Pawel, Tricia Bhatti, Mariarita Santi, Javed Khan, Michael Taylor, Dimiter S. Dimitrov, Crystal Mackall, John M. Maris. GPC2 is an oncogene and immunotherapeutic target in 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 685. doi:10.1158/1538-7445.AM2017-685

Whole Genomic Copy Number Alterations in Circulating Tumor Cells from Men with Abiraterone or Enzalutamide-Resistant Metastatic Castration-Resistant Prostate Cancer.

Purpose: Beyond enumeration, circulating tumor cells (CTCs) can provide genetic information from metastatic cancer that may facilitate a greater understanding of tumor biology and enable a precision medicine approach.Experimental Design: CTCs and paired leukocytes from men with metastatic castration-resistant prostate cancer (mCRPC) were isolated from blood through red cell lysis, CD45 depletion, and flow sorting based on EpCAM/CD45 expression. We next performed whole genomic copy number analysis of CTCs and matched patient leukocytes (germline) using array-based comparative genomic hybridization (aCGH) from 16 men with mCRPC, including longitudinal and sequential aCGH analyses of CTCs in the context of enzalutamide therapy.Results: All patients had mCRPC and primary or acquired resistance to abiraterone acetate or enzalutamide. We compiled copy gains and losses, with a particular focus on those genes highly implicated in mCRPC progression and previously validated as being aberrant in metastatic tissue samples and genomic studies of reference mCRPC datasets. Genomic gains in >25% of CTCs were observed in AR, FOXA1, ABL1, MET, ERG, CDK12, BRD4, and ZFHX3, while common genomic losses involved PTEN, ZFHX3, PDE4DIP, RAF1, and GATA2 Analysis of aCGH in a sample with sequential enzalutamide-resistant visceral progression showed acquired loss of AR amplification concurrent with gain of MYCN, consistent with evolution toward a neuroendocrine-like, AR-independent clone.Conclusions: Genomic analysis of pooled CTCs in men with mCRPC suggests a reproducible, but highly complex molecular profile that includes common aberrations in AR, ERG, c-MET, and PI3K signaling during mCRPC progression, which may be useful for predictive biomarker development. Clin Cancer Res; 23(5); 1346-57. ©2016 AACR.

Heterogeneity of MYCN amplification in neuroblastoma at diagnosis, treatment, relapse, and metastasis.

Amplification of the MYCN gene in neuroblastoma is associated with a poor prognosis and is considered to remain unchanged in post-treatment specimens and metastases. While heterogeneity of MYCN copy number in tumor cells has been reported, serial samples have only been studied in a limited way, and the biologic relevance of this finding is not well understood. We used in situ hybridization on paraffin sections of 102 specimens from 30 patients with MYCN-amplified neuroblastoma to determine MYCN copy number in the primary tumor, pre- and post-treatment, and in metastatic samples. Nineteen cases (63%) showed diffuse MYCN amplification in all samples tested. Nine cases (30%) showed a reduction in MYCN copy number: five cases with diffuse amplification subsequently showed focal amplification, one case with diffuse MYCN amplification showed MYCN gain after treatment, and three focally amplified cases were non-amplified in later specimens. In two cases (7%), focal amplification became diffuse in subsequent samples. Histology was not predictive of the temporal or spatial pattern of MYCN amplification for a particular tumor. If extent of amplification (focal vs. diffuse) is not considered, 26/30 (87%) of cases were consistently MYCN-amplified. However, our data suggest that MYCN status can be heterogeneous between tumor sites, during tumor progression or following treatment, challenging the notion that MYCN copy number does not change for a particular neuroblastoma. Assessing the biologic significance of MYCN heterogeneity will require larger studies of clinically annotated tumor samples, and will depend on interpreting heterogeneity in MYCN status in combination with other genetic changes. © 2016 Wiley Periodicals, Inc.

Detection of MYCN gene amplification in neuroblastic tumors by fluorescence in situ hybridization and its clinical significance

Objective To explore the significance of MYCN gene amplification in children with neuroblastic tumors(NT). Methods The clinicopathological data of 154 cases with NT were reviewed, including general data, classification of pathology, clinical stage and prognosis.MYCN gene amplification was detected by fluorescence in situ hybridization(FISH) and its relationship between pathological characteristics and prognostic significance was analyzed. Results There was 154 cases of NT aged 1 day to 11 years, with a mean age of 26.1 months, and the median age of 20.5 months.Male and female ratio was 1.48∶1.00.According to International Neuroblastoma Staging System(INSS), 20 cases were of stageⅠ(13.0%), 23 cases of stageⅡ (14.9%), 43 cases of stage Ⅲ(27.9%), 64 cases of stage Ⅳ(41.6%) and 4 cases of Ⅳs (2.6%). There were 72 cases(46.8%) with favorable histology, and 82 cases(53.2%) with unfavorable histology.MYCN amplification was found in 20 cases (13.0%) and the signal ratio of MYCN and chromosome 2(CEP2) was 4.08-43.29.One hundred and thirty-four cases of MYCN non-amplification included MYCN gain in 91 cases(68.0%), MYCN negative in 43 cases(32.0%). MYCN expression showed the signi-ficant differences in ages, neuroblastoma type, international neuroblastoma pathology classification(INPC), mitosis kar-yorrhexis index (MKI), and clinical stages(all P 0.05). Of 20 MYCN amplification cases, 4 cases (20.0%) survived and 16 cases (80.0%) died, and the overall survival rate was 20.0%(4/20 cases), with survival time was (17.10±2.24) months; of 134 MYCN non-amplification cases, 96 cases (71.6%) survived and 38 cases (28.4%) died, with survival time of (28.71±1.28)months.Survival analysis showed the cases with MYCN amplification had worse prognosis(χ2=19.596, P<0.05). Conclusions Patients with MYCN amplification had poorer prognosis and lower incidence of MYCN amplification of pediatric NT was found in China. Key words: Neuroblastoma; MYCN gene; Fluorescence in situ hybridization; Child

Abstract A1-59: Multiple mechanisms of MYCN dysregulation in Wilms tumor

Abstract Background and Purpose: Wilms tumor (WT) is the commonest pediatric renal malignancy. A significant minority of cases have mutations in known genes or recurrent copy number aberrations, but few of these have prognostic value. We have previously observed MYCN copy number gain in WT, an aberration associated with poor outcome in several other childhood cancers. We therefore set out to assess the prognostic significance of MYCN gain in a large WT series, and to explore other mechanisms by which the function of this gene may be dysregulated. Methods: MYCN copy number was measured in 293 tumours by Multiplex ligation-dependent probe amplification. Whole exome sequencing (Agilent SureSelect V4+UTR/Illumina HiSeq) was carried out on 51 WT tumour/germline pairs. Sanger sequencing was used to confirm variants of interest and to assess an independent panel of 168 WTs. 65 WTs were profiled on Affymetrix SNP arrays (250K Nsp or SNP 6.0), Illumina HumanMethylation450 arrays, and Illumina HumanHT-12 expression arrays, and additional selected tumors on Illumina HumanCytoSNP-12 arrays. Results: MYCN gain is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. A somatic p.P44L (c.131C&amp;gt;t) mutation was detected in 3/45 exomes that gave informative data at this position, and in 5/168 additional tumors screened for this variant, an overall frequency of 3.8%. MYCN overexpression in high risk tumors was associated with hypomethylation at specific loci. We observed parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumors of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolved over time. In a second bilateral case, we detected MYCN gain as a germline aberration. Conclusions: MYCN gain is associated with poor outcome and anaplastic histology in WT. MYCN also appears to be disrupted by other mechanisms in WT, including DNA hypomethylation and a recurrent potential gain of function mutation, P44L. The discovery of a germline aberration suggests that MYCN dysregulation may contribute to tumor predisposition. Citation Format: Richard D. Williams, Tasnim Chagtai, Marisa Alcaide-German, John Apps, Jenny Wegert, Sergey Popov, Gordan Vujanic, Harm van Tinteren, Marry M. van den Heuvel-Eibrink, Marcel Kool, David Gisselsson, Norbert Graf, Manfred Gessler, Kathy Pritchard-Jones. Multiple mechanisms of MYCN dysregulation in Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-59.

Genetic abnormalities in adolescents and young adults with neuroblastoma: A report from the Italian Neuroblastoma group.

Less than 5% of neuroblastomas (NB) occur in adolescents and young adults (AYA), in whom the disease has an indolent and fatal course. We studied the genomic profile and histological characteristics of 34 NBs from AYA patients enrolled in the Italian Neuroblastoma Registry (INBR) between 1979 and 2009. Disease was disseminated in 20 patients and localized in 14; 30/34 tumors were classified as NB and 4/34 as nodular ganglioneuroblastoma (nGNB). Segmental Chromosome Aberrations (SCAs) were observed in 29 tumors (85%) namely 1p imbalance (58%), 17q gain (52%), 9p loss (32%), 11q loss (30%), 1q gain (17%), 7q gain (17%), 2p gain (14%), 3p loss (14%), and 4p loss (7%). MYCN amplification and MYCN gain were detected in 3 (10%) and 2 cases (7%) respectively. An anaplastic lymphoma receptor tyrosine kinase (ALK) gene mutation study on the available cases from this cohort revealed 4/25 (16%) mutated cases. In parallel, alpha thalassaemia/mental retardation syndrome X linked (ATRX) gene mutations were also sought, a novel mutation being detected in 1/21 (4,7%) cases. This study confirmed the low incidence of MYCN amplification in AYA and recorded a high frequency of 17q gain and 9p and 11q loss independently from the stage of the disease. The presence of 1q gain, which identifies patients with particularly aggressive disease, relapse and poor survival, was also detected. Furthermore, the frequency of ALK mutations suggests that a target-based therapy with ALK inhibitors might be effective in this subset of patients.

The neuroblastoma and ganglion components of nodular ganglioneuroblastoma are genetically similar: evidence against separate clonal origins

Nodular ganglioneuroblastoma is characterized by a macroscopic nodule of neuroblastoma within a ganglioneuromatous component. These two components have been considered to originate from separate clones, with the neuroblastoma clone accounting for the clinical behavior of nodular ganglioneuroblastoma. In order to investigate the clonal origin of the cellular components (neuroblasts, ganglion cells, and Schwann cells) of nodular ganglioneuroblastoma, paraffin-embedded tumor samples from eight cases were analyzed by single nucleotide polymorphism array and in situ hybridization. DNA was extracted separately from neuroblastomatous and ganglioneuromatous areas. By in situ hybridization, MYCN gain (4–10 gene copies/nucleus) was detected in 7/8 neuroblastoma samples. In ganglioneuromatous regions, gains were also detected in ganglion cells but not in Schwann cells. Single-nucleotide polymorphism array studies identified chromosome losses (11q and 14q) and gains (12, 13q, 17q and 18q) in the neuroblastoma component, whereas the ganglioneuromatous component showed fewer or no genetic alterations. There were no unique copy number changes distinguishing nodular ganglioneuroblastoma from other subtypes of neuroblastoma. By in situ hybridization, ganglion cells but not Schwann cells showed the same alterations detected in neuroblasts. Thus, neuroblasts and ganglion cells in nodular ganglioneuroblastoma are genetically related and may arise from the same clone. In contrast, the Schwann cells have a different origin and may be derived from a non-neoplastic neural crest precursor. Our results suggest that the clinical behavior of nodular ganglioneuroblastoma cannot be explained by the presence of separate clones with distinct genetic signatures.