Positive Neuroblastoma Research Articles

HGG-48. THE BLOOD-BASED TELOMERIC DNA C-CIRCLE ASSAY CAN DETECT ALTERNATIVE LENGTHENING OF TELOMERES (ALT) IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND High-grade gliomas (HGG) comprise ~10% of pediatric brain tumors; 40% of pediatric HGG use the alternative lengthening of telomeres (ALT) mechanism to maintain replicative immortality. ALT cancers share a unique biology providing potential therapeutic targets. Extrachromosomal telomere DNA repeats, termed C-circles, can be detected by a unique PCR assay, providing a sensitive and specific biomarker for ALT cancers. C-circles have been detected in ALT tumors and serum of ALT patients. We have adapted the C-circle assay (CCA) to provide a sensitive and specific assay with cfDNA in patient plasma. Here we determined if cfDNA in plasma can be used to detect patients with ALT-positive HGG. METHODS Frozen plasma samples were collected in Streck tubes at time of surgery prior to resection from patients with HGG. DNA was extracted and quantified. Isothermal rolling C-circle amplification and real-time telomere PCR was performed followed by analysis. The C-circle positive neuroblastoma cell line, CHLA-90, and C-circle negative neuroblastoma cell line, CHLA-20, served as the positive and negative controls respectively. CCA values in plasma of 2 and above indicate positivity for C-circles. RESULTS Plasma samples were analyzed by CCA from three pediatric patients whose brain tumors were previously biopsied and identified as ATRX mutation positive. ATRX mutation indicates ALT however, not all ALT cancers have ATRX mutations. All plasma samples were above the 2.0 cutoff at 3.37, 3.13, and 4.59 and thus classified as C-circle positive. CONCLUSIONS This preliminary cohort shows that the CCA has potential for identifying ALT pediatric brain tumors using cfDNA from plasma. As novel therapies effective against ALT HGG are developed, the plasma CCA may allow accelerated initiation of neoadjuvant therapy to shrink tumor prior to surgical resection and lessen or prevent the need for tumor biopsy. Expansion of the initial test cohort is underway. Supported by NCI UO1 CA63988.

CD99 and NKX2.2 positive neuroblastoma diagnosed on cytology: A potential diagnostic pitfall and necessity of pathological evaluation of the primary site.

Immunohistochemistry plays a vital role in the diagnosis of small round cell tumors. CD99 immunonegativity is one of the features, which helps in distinguishing neuroblastoma from other small round cell tumors. NKX2.2 is a specific marker of Ewing sarcoma, which is a differential for poorly differentiated neuroblastoma. Here, we present a case of metastatic neuroblastoma showing immunoreactivity for both CD99 and NKX2.2 on cytology of the metastatic site causing diagnostic dilemma. Biopsy study of the adrenal lesion revealed presence of differentiating cells and neuropil, highlighting the importance of evaluation of the primary site and limitation of cytology.

Identification of hub genes and their correlation with infiltration of immune cells in MYCN positive neuroblastoma based on WGCNA and LASSO algorithm

BackgroundThe prognosis of MYCN positive NB is poor, and there is no targeted drug for N-myc at present. This study aims to screen out hub genes closely related to MYCN, analyze the relationship between hub genes and NB microenvironment, and provide basis for molecular targeted therapy of MYCN positive NB.MethodsWe combined the microarray data of GSE45547 (n=649) and GSE49710 (n=498), screened the DEGs between MYCN positive (n=185) and MYCN negative NB (n=951), performed WGCNA, Lasso regression and Roc analyses on the merged matrix, and obtained the hub genes related to MYCN in the training group. We performed ssGSEA on the experimental group to calculate the infiltration level of 28 kinds of immune cells in each sample, compared the differences of immune cell infiltration between MYCN positive and MYCN negative group. The influences of hub genes on the distribution of each immune cell were also analyzed by ssGSEA. The expression differences of the three hub genes were verified in the E-MTAB-8248 cohort (n=223), and the correlation between hub genes and prognosis of NB was calculated by Kaplan-Meier method in GSE62564 (n=498) and the validation group. We also verified the expression differences of hub genes by qRT-PCR in SK-N-BE(2), SKNDZ, Kelly and SH-SY5Y cell lines.ResultsHere were 880 DEGs including 420 upregulated and 460 downregulated genes in MYCN positive NB in the training group. Overlap of the DEGs and WGCNA networks identified four shared genes, namely, ZNF695, CHEK1, C15ORF42 and EXO1, as candidate hub genes in MYCN positive NB. Three core genes, ZNF695, CHEK1 and C15ORF42, were finally identified by Lasso regression and Roc analyses. ZNF695, CHEK1 and C15ORF42 were highly expressed in MYCN positive NB tissues and cell lines. These three genes were closely related to the prognosis of children with NB. Except that Activated CD4 T cell and Type2 T helper cell increased, the infiltration levels of the other 26 cells decreased significantly in MYCN positive NB tissues. The infiltration levels of Type2 T helper cell and Activated CD4 T cell were also significantly positively correlated with the expression levels of the three hub genes.Conclusion ZNF695, CHEK1 and C15ORF42 are highly expressed in MYCN positive NB, and their expression levels are negatively correlated with the prognosis of children with NB. The infiltration levels of Activated CD4 T cell and Type2 T helper cell increased in the microenvironment of MYCN positive NB and were significantly positively correlated with the expression levels of the three hub genes. The results of this study provide that ZNF695, CHEK1 and C15ORF42 may be potential prognostic markers and immunotherapy targets for MYCN positive NB.

Abstract 1498: Nanobody-based CAR T cells targeting B7-H3 in pancreatic cancer

Abstract Pancreatic cancer is a common cause of cancer-related mortality worldwide with a poor 5-year survival rate. Adoptive transfer of T cells engineered with chimeric antigen receptors (CAR T cells) have shown promising potency in hematological malignancies, but it is more challenging to apply CAR T cells in the treatment of solid tumors. One of the major barriers is the lack of appropriate therapeutic targets. B7-H3 (CD276) is an emerging and promising pan-cancer target that is overexpressed in multiple solid tumors including pancreatic ductal adenocarcinoma (PDAC) and neuroblastoma (NB) but limited in normal tissues. Interestingly, it is highly expressed in not only tumor cells but also tumor-associated myeloid cells in the tumor microenvironment. In this study, we isolated a panel of novel B7-H3 specific nanobodies from our camel VHH single domain phage libraries. These nanobodies bind to human B7-H3-expressing tumor cell lines and cross-react with B7-H3 of different species. Notably, three nanobody-based CAR T cells (B12, C4 and G8) demonstrate potent cytolytic effects on B7-H3 positive PDAC and NB cell lines in vitro. In particular, CAR T cells have been tested in a metastatic PDAC model and a NB model in mice, showing that C4 and B12 nanobody-based CAR T cells have potent antitumor efficacy without evident side effects. Therefore, we conclude that our new B7-H3 targeted nanobody-based CAR T cells, in particular C4 and B12 derived CAR T cells, are promising for treating solid tumors such as pancreatic cancer and other deadly solid tumors. Citation Format: Dan Li, RuiXue Wang, Tianyuzhou Liang, Brad St. Croix, Mitchell Ho. Nanobody-based CAR T cells targeting B7-H3 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1498.

A case of synchronous malignancy with novel missense mutation in a child: Is this Li-Fraumeni syndrome or a novel case masquerading as Li-Fraumeni syndrome??

AbstractWe report the case of an 11-month-old child who presented with a change in voice, increased weight, and hirsutism, who was also found to have elevated levels of serum cortisol and testosterone, showing three synchronous malignancies in the liver, left adrenal gland, and posterior mediastinum. Clinical exome sequencing report revealed germline TP53 (P177A) and MLH3 (V741P) mutations with NMYC positive neuroblastoma. At the outset, this may look like a Li-Fraumeni syndrome (LFS) with TP53 germline mutation but lacks other features to be termed as LFS or Li-Fraumeni-like syndrome. The gene mutation variant found in this case (P177A) is a novel missense mutation which has never been reported, and the MLH3 gene mutation variant V741P has not been previously associated with any of the malignancies seen in this child.

Association Between Patient Characteristics and Diet Profile with Kirsten rat sarcoma Kras and Neuroblastoma rat sarcoma (Nras) Gene Mutation in Colorectal Cancer

Background: Colorectal cancer is the third most common cancer according to American Cancer Society. It is also the third most common cause of death in men and women in US. Colorectal cancer encompasses 5% of all cancer and 29% of gastrointestinal cancer with men and women ratio is about 3:1. More than 1/3 of colorectal cancer occur below the age of 45 years. Mutation in Kirsten rat sarcoma (KRAS) gene was found in 30-50% of colorectal cancer in which it was suggested to associated with increase proliferation and decrease apoptosis. This study aimed to analyze the association between diet profile and KRAS gene mutation.Method: This study was a cross sectional study. Data was collected from medical records of colorectal cancer patient in Dr. Saiful Anwar General Hospital, which included KRAS gene mutation analysis.Results: There were 12 subjetcs included in this study. Four subjects (33.3%) had gene mutation with 3 subjects (75%) had positive KRAS mutation and 1 subjects (25%) had positive Neuroblastoma rat sarcoma (NRAS) mutation. In non-mutation group, it had been found a higher frequency of green leaf vegetables diet, in comparison with mutation group (p = 0.023). There was positive correlation between green leaf vegetables diet with gene mutation.Conclusion: Mutation of KRAS and NRAS mutation in colorectal carcinoma were found in 33.3% of subjects. Data analysis showed positive association between low green leaf vegetables diet with KRAS and NRAS mutation.

Alectinib, an Anaplastic Lymphoma Kinase Inhibitor, Abolishes ALK Activity and Growth in ALK-Positive Neuroblastoma Cells.

Oncogenic receptor tyrosine kinases including anaplastic lymphoma kinase (ALK) are implicated in numerous solid and hematologic cancers. ALK mutations are reported in an estimated 9% of neuroblastoma and recent reports indicate that the percentage of ALK-positive cases increases in the relapsed patient population. Initial clinical trial results have shown that it is difficult to inhibit growth of ALK positive neuroblastoma with crizotinib, motivating investigation of next generation ALK inhibitors with higher affinity for ALK. Here, alectinib, a potent next generation ALK inhibitor with antitumor activity was investigated in ALK-driven neuroblastoma models. Employing neuroblastoma cell lines and mouse xenografts we show a clear and efficient inhibition of ALK activity by alectinib. Inhibition of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. The results suggest that alectinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma and should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Abstract 1437: Improving efficacy and accuracy of CAR-T cells for neuroblastoma

Abstract Background: Anti-disialoganglioside (GD2) specific CAR T-cell therapy has shown limited efficacy in children with recurrent/refractory neuroblastoma (NBL). A recent pre-clinical study demonstrated significant neurotoxicity in mice treated with CAR T-cells bearing a high-affinity anti-GD2 short chain variable fragments (scFv; Richman S et al. 2017). To devise a safe and more efficacious CAR T-cells, we developed a gated CAR T-cell system utilizing the Syn-Notch technology. Methods: GD2 and CD19 specific Syn-Notch gated systems were constructed by linking corresponding scFv to the Notch core followed by Gal4Vp64. Upon target recognition, Gal4VP64-dependent UAS element would initiate expression of the B7H3-BBZ CAR. Reporter activity of CD19 or GD2 Syn-Notch systems were monitored using flow cytometry after exposure to CD19+ transduced CHLA255 NBL cell line. In vitro cytotoxicity was assessed using DIMSCAN and xCELLigence impedance assays at varying Effector: Target ratios. NSG mice with established metastatic CHLA255(GD2+CD19+) NBL were treated with 1x10^7 transduced and control T cells and monitored for disease burden. Results and discussion: Jurkat and human primary T cells were successfully transduced with the Syn-Notch system. GD2-Notch-Gal4VP64 and CD19-Notch-Gal4VP64 T-cells effectively activated downstream reporter in exposure to CHLA255(GD2+CD19+) within 6 hours of exposure but not in the presence of NBL cells lacking GD2 or CD19. The GD2 system exhibited greater leakiness than the CD19 counterpart. GD2- Syn-Notch-B7H3-BBZ or CD19- Syn-Notch-B7H3-BBZ CAR T-cells demonstrated statistically significant cytotoxicity against CHLA255(GD2+CD19+) NBL cell line. Preliminary in vivo data also show the anti-tumor effect of these gated CAR systems compared to mock-transduced T-cells. Conclusions: We have successfully established a novel GD2-B7H3 gated system using the Syn-Notch technology, and demonstrate successful specificity to both antigen targets. This system increases the specificity of CAR T-cells against GD2 and B7H3 positive neuroblastoma tumors and likely minimize GD2-induced toxicity. These gated CARs show great promise for establishing immunotherapies that maximize tumor specificity while minimizing toxicity. Citation Format: Babak Moghimi, Sakunthala Muthugounder, Shahab Asgharzadeh. Improving efficacy and accuracy of CAR-T cells for neuroblastoma [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 1437.

Modulation of anti-tumour activity of ex vivo expanded NK and GD2CAR-NK cells against neuroblastoma

Background & Aim Chimeric antigen receptor engineered T cells (CAR T) have shown clinical success especially in acute lymphoblastic leukemia. Innate Natural Killer (NK) cells bear functional resemblance to T cells, but display a safer cytokine profile, immediate availability and no risk of GvHD. CAR NK are thus an attractive alternative to CAR T, especially in an allogeneic setting. Our study aims to investigate the cytolytic activity of NK and GD2CAR-NK cells against GD2 positive neuroblastoma (NB), either alone or in combination with histone deacetylase inhibitor (HDACi) and Programmed death 1 (PD-1) blocker. Methods, Results & Conclusion We have shown NK cells expanded from mononuclear cells by co-culture with irradiated K562-mbIL15-41BBL cells exhibited robust cytotoxicity against NB cells. HDACi (selective Class I inhibitor entinostat and pan-inhibitor panobinostat) up-regulated surface expression of NKG2D ligands MIC A/B & ULBPs on NB cells and significantly increased NK-mediated killing in NB cell lines and patient-derived-xenograft (PDX) samples. Immune checkpoint blockade via neutralizing antibodies against PD1/PDL1 axis (upregulated during NB and NK co-culture), when used in conjunction with HDACi, further improved efficiency of NK-mediated killing. In NB subcutaneous and metastatic xenograft model, adoptive NK cell therapy significantly prolonged event free survival, an effect further enhanced by treatment with both HDACi and PD1 blocker. Retrovirally transduced GD2CAR-NK cells significantly increased killing against GD2-positive NB cells as compared to control NK cells. Live cell imaging showed robust infiltration of GD2CAR-NK cells into 3D tumor spheroids, significantly reduced growth rate and able to destroy tumor spheroid. In a metastatic xenograft model, GD2CAR-NK treated mice showed significantly lower bioluminescence signal 11 days post tumor inoculation compared to control NK cells. However, although lower signal persisted in GD2CAR-NK group, significance was lost by day 18. Persistence of NK cells in mice was detected for one time point and no expansion observed. Taken together, our data suggest that NK cells mediate efficient killing of NB cells, enhanced by use of HDACi and PD1/PD-L1 blocker. GD2CAR-NK further enhance killing of target cells in vitro and although transiently, slowed tumor progression in vivo. Further investigation to improve transduction efficiency and NK/GD2CAR-NK cell expansion and persistence, with aim to enhance efficacy of NK/GD2CAR-NK cell therapy in vivo.

Association between image-defined risk factors and neuroblastoma outcomes

BackgroundThe current neuroblastoma (NBL) staging system employs image-defined risk factors (IDRFs) to assess numerous anatomic features, but the impact of IDRFs on surgical and oncologic outcomes is unclear. MethodsThe Vanderbilt Cancer Registry identified children treated for NBL from 2002 to 2017. Tumor volume (TV) and IDRFs were measured radiographically at diagnosis and before resection. Perioperative and oncologic outcomes were evaluated. ResultsAt diagnosis of 106 NBL, 61% were IDRF positive. MYCN-amplified and undifferentiated NBL had more IDRFs than nonamplified and more differentiated tumors (p = 0.001 and p = 0.01). Of 86 NBLs resected, 43% were IDRF positive, which associated with higher stage, risk, and TV (each p < 0.001). The presence of IDRF at resection was also associated with increased blood loss (p < 0.001), longer operating times (p < 0.001), greater incidence of intraoperative complications (p = 0.03), more frequent ICU admissions postoperatively (p < 0.001), and longer hospital stays (p < 0.001). IDRF negative and positive tumors did not have significantly different rates of gross total resection (p = 0.2). Five-year relapse-free and overall survival was similar for IDRF negative and positive NBL (p = 0.9 and p = 0.8). ConclusionsIDRFs at diagnosis were associated with larger, less differentiated, advanced stage, and higher risk NBL and at resection with increased operative difficulty and perioperative morbidity. However, the frequency of gross total resection and patient survival after resection were not associated with the presence of IDRFs. Type of studyRetrospective cohort study. Level of evidenceLevel III.

Immune landscape and in vivo immunogenicity of NY-ESO-1 tumor antigen in advanced neuroblastoma patients

BackgroundIndirect evidence suggesting the immunosensitivity/immunogenicity of neuroblastoma is accumulating. The aims of this study were to investigate the immune landscape of neuroblastoma and to evaluate the in vivo immunogenicity of the NY-ESO-1 tumor antigen in advanced neuroblastoma patients.MethodsThe immune infiltrating cells of the NY-ESO-1+ tumors from three HLA*A201 patients with metastatic neuroblastoma who relapsed after conventional treatments were evaluated by immunohistochemistry. The patients were vaccinated with the HLA-A*0201-restricted peptide NY-ESO-1157-165(V). The peptide was emulsified in Montanide ISA51 and given subcutaneously in a phase I pilot study. The immunogenicity of NY-ESO-1 antigen was evaluated by monitoring mononuclear cells in patient peripheral blood, pre- and post-vaccine, by short-term in vitro sensitization, HLA-multimer staining and IFN-γ ELISpot analysis.ResultsBoth CD3 T cells and CD163 myeloid cells were present in pre-vaccine tumors and PD-1 and PD-L1 expression was mainly found in the immune infiltrate. Despite the advanced stage of the disease, the vaccination induced systemic NY-ESO-1 specific CD8 T cells releasing IFN-γ in response to activation with the NY-ESO-1 peptide and an HLA-A2 positive neuroblastoma cell line.ConclusionsOur results indicate that vaccination with a tumor-associated peptide is able to boost NY-ESO-1-specific, functionally active T cells in advanced neuroblastoma patients with lymphocyte infiltration in their pre-vaccine tumors.Trial registrationEudraCT #2006–002859-33.

PO-124 PTTG and PBF functionally interact with P53 and predict overall survival in head and neck cancer

IntroductionReceptor tyrosine kinases (RTKs) play important roles in cellular proliferation and differentiation. In addition, RTKs reveal oncogenic potential when their kinase activities are constitutively enhanced by point mutation, amplification, or rearrangement of the corresponding genes. We are investigating ALK (Anaplastic Lymphoma Kinase) tyrosine kinase in neuroblastoma (NB) - the most frequently occurring extracranial tumour in children, where ALK activating mutations are found.In this study we performed phosphoproteomic and a gene signature profiling of ALK activity from several different neuroblastoma cells comparing treatment with the first and third generations ALK inhibitors.Material and methodsCrizotinib was one of the first ALK TKIs to be described; however the response in patients with ALK positive NB was poor. In contrast, robust and sustained clinical responses we seen in ALK rearranged IMT. Lorlatinib is a third generation ALK TKI with very positive responses in NSCLC. We treated two ALK addicted (CLB-BAR, CLB-GE) and one ALK non-addicted (SK-N-AS) NB cell lines with either crizotinib or lorlatinib and performed phosphoproteomic and RNAseq profiling to identify common targets for future combinational treatment for patients.Results and discussionsProfiling of ALK addicted CLB-BAR and CLB-GE cell lines led to the identification of 3345 and 2252 phosphoproteins, respectively. Phosphorylation of the ALK receptor itself was decreased in both lines upon treatment with crizotinib or lorlatinib. Parallel RNAseq profiling was performed at 24 hours. Comparison with non-treated controls revealed 19 232 differently expressed genes. In ALK addicted cells phosphoproteomic analysis revealed more than 50 proteins that were dephosphorylated upon treatment with ALK TKIs. More than 400 genes were downregulated, and more than 600 were upregulated, upon treatment with either crizotinib or lorlatinib. As an outcome we obtained a list of predicted targets for further analysis. We focused on validation of downstream signalling molecules such as: FOXO3a/4, DUSP4 and ETV3/4.ConclusionIn this study we established and compared phosphoproteomic and gene signature profiles of ALK activity in NB cells employing first and third generation ALK TKIs. This analysis has unveiled a number of leads for novel combinatorial treatment strategies for NB patients as well as an increased understanding ALK dependant signalling processes.

PO-314 ALK inhibitor, ceritinib, abrogates activation of the novel ALK-I1171T mutation in neuroblastoma

IntroductionMutations in the kinase domain of Anaplastic Lymphoma Kinase (ALK) are undoubtedly implicated in the pathogenesis of the childhood cancer, neuroblastoma. However, clinical response of ALK positive neuroblastoma patients to the first generation ALK inhibitor has been rather disappointing.Here we report the appearance of a novel ALK mutation in neuroblastoma together with other chromosomal aberrations that mediate neuroblastoma initiation and progression.Material and methodsGenomic tumour DNA from biopsy samples were extracted and exome sequencing was performed through paired-end sequencing on Illumina plateforms.The novel ALK-I1171T mutant was biochemically analysed by western blot and neurite-outgrowth assay in PC12 cells, and foci formation assay in NIH3T3 cells.Results and discussionsAnalyses of genomic tumour DNA from biopsy samples initially showed an 11q deletion, 17q gain with a mutation of the ALK gene at protein position 1171, which mediate an amino acid change from isoleucine to threonine. We show that mutation of I1171 to threonine generates a potent gain-of-function mutant, as observed in two independent systems. Firstly, in PC12 cell lines expressing ALK-I1171T display ligand independent activation of ALK, neurite outgrowth and further downstream signalling activation. Secondly, ALK-I1171T meditate foci formation in a NIH3T3 transformation analysis. Finally, pharmacological inhibition of ALK-I1171T employing ceritinib, an FDA approved ALK inhibitor show 14-fold better ability to abrogate ALK-I1171T compared with crizotinib.ConclusionThis study suggests that ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

Abstract 4130: TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma

Abstract Centrosomes are the major microtubule-organizing centers in animal cells. Centrosomes catalyze microtubule assembly to accelerate formation of the bipolar mitotic spindle that segregates chromosomes during cell division. To analyze the effect of centrosome removal in normal and cancer cells, we developed centrinone, a potent, specific and cellularly-active inhibitor of the protein kinase polo-like kinase 4 (Plk4), which is essential for centrosome duplication (Wong et al., 2015, Science 348:1155-60). Centrosome-less cells take longer to execute mitosis, and trigger a mitotic stress signaling pathway that results in p53 stabilization and apoptosis and/or senescence of the resulting daughter cells. Failure to form a spindle can also lead to apoptosis during mitosis or exit from mitosis without chromosome segregation (mitotic slippage). In a genome-wide CRISPR/Cas9 loss-of-function screen for resistance to centrinone, we had previously shown that loss of the ubiquitin ligase TRIM37 makes spindle assembly more robust to centrosome removal (Meitinger et al., 2016, J Cell Biol. 214:155-66), raising the possibility that high levels of TRIM37 might increase sensitivity to centrosome removal. Here, we describe the use of single-cell live imaging assays to identify determinants of vulnerability of cancer cells to centrosome removal. Among a panel of 37 tested cell lines, neuroblastoma-derived cell lines were the most sensitive to centrosome removal, exhibiting high levels of apoptosis and rapid loss of viability following treatment with centrinone. We found that sensitivity to centrosome removal correlated with TRIM37 expression levels. Upon centrosome depletion, cells with high TRIM37 levels, particularly neuroblastoma cells, exhibited greatly increased mitotic duration and frequency of mitotic failure. p53 positive neuroblastoma cell lines exhibited apoptosis resulting from mitotic stress-mediated p53 activation and from mitotic failure. Surprisingly, p53 negative neuroblastoma cell lines, which are unable to sense mitotic stress, were also highly sensitive to centrosome removal, with cell death in this case resulting from high rates of mitotic failure. TRIM37 deletion restored the ability of cells to proliferate in centrinone. Thus, centrosome removal via Plk4 inhibition appears to be a promising strategy for therapeutic treatment of neuroblastomas and potentially other cancers with high levels of TRIM37 expression. Citation Format: Franz Meitinger, Robert L. Davis, Ruth Kabeche, John V. Anzola, Yao Liang Wong, Andrew K. Shiau, Arshad Desai, Karen Oegema. TRIM37 expression levels dictate susceptibility to centrosome removal, supporting Plk4 inhibition as a potential new strategy for targeting neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4130.

Abstract 4876: Low frequent ALK hotspot mutations in neuroblastoma tumors detected by ultra deep sequencing: Implications for ALK inhibitor treatment

Abstract Background The pediatric cancer neuroblastoma (NB) is heterogeneous in terms of both genotype and clinical behavior. NB patients show complex patterns of genetic abnormalities, which may include amplification, translocation or oncogenic mutations of ALK kinase in sporadic and familial cases. Three hot spot residues (F1174, F1245, and R1275) localized within the kinase domain of ALK, accounts for 85% of mutant ALK in NB. In this study we used ultra deep sequencing for sensitive detection of ALK mutations. Methods To detect the sub-clonal ALK mutations in 105 neuroblastoma tumors that can remain undetected using Sanger chemistry, we used two different approaches; hybridization-based Haloplex kit (Agilent) and PCR-based amplicon sequencing, both sequenced on the MiSeq Illumina platform. Coverage, uniformity and variant calling ability showed similar performance between both methods. Findings ALK mutations were detected in 14/105 patients (13%) whereof six with allele frequency below 20%. At the I1171 locus an ALK mutation was detected in one case with variant allele frequency as low as 2.6%, the lowest level detected in our cohort. Mutations at the F1174 hotspot were observed in nine cases: six cases with F1174L, while the remaining three showed F1174C, F1174S and F1174I substitutions, with mutated allele fractions ranging from 14% to 60%. Interestingly the patient harboring F1174S mutation with 58% frequency, in parallel contained a small sub-clone of F1174I mutation with 8% frequency of the mutated allele. F1245-codon alterations were detected in three cases: two F1245I mutations and one F1245C mutation, with frequencies of 14%, 51% and 52%, respectively. A single case showed a L1240V variant with a mutated allele fraction at 57%. Sanger sequencing of all ALK mutated samples could confirm ALK status for all samples with variant allele fraction above 15%. Because of the limits of detection, 4 out of 6 samples with a mutated allele fraction below 15% would have gone undetected relying on Sanger sequencing. Our results fail to reveal a distinct mutation spectrum in relation to neuroblastoma genomic subtypes. Moreover, a variety of neuroblastic tumours, from benign ganglioneuroma to very aggressive metastatic neuroblastoma, could be observed in the context of ALK mutation. Interpretation These results indicate that, in neuroblastoma, ALK mutations can be present at sub-clonal level with the possibility of subsequent clonal expansion at the time of progression. Tyrosine kinase inhibitors have become the gold standard therapy in treating ALK positive NBs, making diagnostic high sensitive detection of ALK mutations a necessary step in identifying optimal treatment modalities. This study is of utmost importance in clinical practice, highlighting the potential of NGS and the importance of serial samplings for therapeutic decisions. Citation Format: Susanne M. Fransson, Niloufar Javanmardi, Rose-Marie Sjoberg, Per Kogner, Tommy Martinsson. Low frequent ALK hotspot mutations in neuroblastoma tumors detected by ultra deep sequencing: Implications for ALK inhibitor treatment [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 4876. doi:10.1158/1538-7445.AM2017-4876

Abstract 4993: Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma

Abstract Background: Gangliosides such as GD2, GD3 and GM2 are promising targets for antibody-mediated cancer therapy since they are expressed at high levels on the surface of several cancers, including neuroblastomas, sarcomas and melanomas. Anti-GD2 monoclonal antibodies have shown promising clinical outcomes in neuroblastoma and a chimeric anti-GD2 antibody (Unituxin™) was recently approved by FDA. However, murine-derived antibodies discovered so far show adverse effects that limit their clinical utility. Fully human antibodies derived from immunized patients might be able to overcome these limitations, since epitope specificity has evolved in the human tissue environment. Here we describe the characterization and functional evaluation of two potential development candidates. Methods: PBMCs from patients immunized with GD2 lactone-keyhole limpet hemocyanin (GD2L-KLH) conjugate vaccine were utilized to isolate fully human monoclonal antibodies (humAbs). HumAbs were selected for 1) specificity against GD2 initially by ELISA and cell surface binding by flow cytometry 2) by glycan array and affinity assays and 3) by effector functions in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) assays. The therapeutic potential of lead antibodies was further evaluated in xenograft models in SCID mice using GD2 positive CHLA255 neuroblastoma as well as with TC71 and SaoS2 sarcoma cell lines. Results: Antibodies that bind specific to purified GD2 by ELISA were expressed as recombinant antibodies in CHO cells. Many recovered antibodies showed cross-reactivity with several gangliosides in ELISA assays. Two antibodies with high specificity for GD2, 1B7 and 31F9 were selected for further studies. Binding to native antigen expressed on the cell surface of different cell lines was confirmed by FACS analysis. 31F9 was monospecific for GD2 (affinity by Surface Plasmon Resonance was ∼4 nM) while 1B7 showed dual specificity for GD2 and GM2 with affinities of ∼1 nM for GD2 and ∼ 370 nM for GM2, respectively. These two antibodies induced CDC ranged between 35% and 80%, and ADCC with human PBMCs between 40% and 65% on CHLA255, TC71 and SaoS2 cell lines. Survival following IV or SC challenge with SaoS2 and TC71 cells (respectively) was at least doubled following six IP treatments with 200 mcg of either 1B7 or 31F9 twice weekly. Inhibition of CHLA255 tumor growth was less dramatic. Conclusions: Vaccinated patients produce antigen-specific IgG antibodies that were further analyzed on the molecular level. Antibodies with single and dual specificity (GD2 and GM2) were recovered and shown to be very active in functional assays. Based on these favorable in vitro and in vivo studies, 1B7 and 31F9 humAbs merit further development efforts to evaluate potential utility for treatment of GD2 positive cancers. Citation Format: Govind Ragupathi, Xiaohong Wu, Ritsuko Sawada, Philip O. Livingston, Wolfgang W. Scholz. Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4993.

Abstract 2303: The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients

Abstract Background: Chimeric antigen receptor (CAR)-engineered T cells have demonstrated potent clinical efficacy in patients with B cell lymphoma. However, the application of CAR-T cell therapy targeting other solid tumors has been limited. The GD2 gangliosides are sialic acid-containing glycosphingolipids that play a role in signal transduction and cell-cell recognition. 1st generation gangliosides GD2 chimeric antigen receptor T cells in some patients with refractory neuroblastoma already studied in a phase I clinical trials, indicate that T cells expressing 1st generation anti-GD2 chimeric antigen receptors were safe and mediated modest antitumor activity. In a phase I study, the murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, was administered intravenously to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma. Antitumor responses occurred in 7 of 17 patients. However, the expression of the gangliosides GD2 in Asian melanoma populations has not been reported. Methods: This study involved tumor samples from 228 melanoma patients (129 acral melanomas, 65 mucosal melanomas, 13 melanomas on skin with chronic sun-induced damage, 21 melanomas on skin without chronic sun-induced damage) in Peking University Cancer Hospital &amp; Institute. Clinical data were collected. Immunohistochemistry assays using antibodies against gangliosides GD2 were performed on formalin-fixed, paraffin-embedded specimens. The ability of gangliosides GD2 chimeric antigen receptor T cells to kill gangliosides GD2+ melanoma cell was evaluated by MTT in vitro. Results: Among the 228 samples,40.3% of the cases(92/228) demonstrated positive staining with gangliosides GD2. Expression of gangliosides GD2 were relatively more frequent in acral (48.1%) and mucosal (36.9%) melanomas than in CSD (15.4%) and Non-CSD (19.0%) melanomas. The median survival time for patients with expression of gangliosides GD2 was significantly shorter than patients with absence of gangliosides GD2 expression (21.4 vs 57.6 months, P&amp;lt;0.05). Statistical differences were found between primary melanomas with metastatic melanomas (34.9% vs 53.0%, P&amp;lt;0.05). In addition, the gangliosides GD2 chimeric antigen receptor T cells exhibited specific lysis of melanoma cell with gangliosides GD2 expression in vitro experiment. Conclusion: These data demonstrate the expression of ganglioside GD2 are more frequent in acral and mucosal melanomas than in cutaneous melanomas in Chinese patients. The higher expression of gangliosides GD2 on metastatic melanomas compared to primary melanomas was in conformity with the viewpoint that GD2 expression was related to increased metastatic potential. Gangliosides GD2 chimeric antigen receptor T cells represents a clinically appealing treatment strategy for melanoma patients with ganglioside GD2 expression. Citation Format: Yan Kong, Jiayi Yu, Lu Si, Zhihong Chi, Chuanliang Cui, Xinan Sheng, Jun Guo. The expression of gangliosides GD2 is associated with worse prognosis in melanoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2303.

657 - Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK positive neuroblastoma cells, Drosophila and mice

657 - Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK positive neuroblastoma cells, Drosophila and mice

Abstract B12: The ALK inhibitor PF-06463922 shows significant response as a single agent in ALK/MYCN driven models of neuroblastoma

Abstract ALK inhibitors such as the ALK/MET/ROS1 inhibitor crizotinib (Xalkori) have shown clinical efficacy in a number of tumour types. However, in ALK positive neuroblastoma treatment with the ALK inhibitor crizotinib has proved more difficult, highlighting the exploration of new drugs as a clinical priority. A recent report of an increased percentage of ALK positive cases in the relapsed neuroblastoma patient population, together with the increased repertoire of ALK inhibitors now available, led to the investigation of alternative ALK inhibitors with potential for use in treatment of neuroblastoma. Here we report an investigation of the activity of a next generation ALK inhibitor in a range of in vitro and pre-clinical ALK driven neuroblastoma models. Initially PF-06463922 was tested in various neuroblastoma cell lines and a range of gain-of-function ALK neuroblastoma mutations were subsequently analyzed in more detail in engineered Ba/F3 and PC12 cell models and by in vitro kinase assays, comparing the effect of PF-06463922 in abrogating cell growth and induced pharmacodynamics markers of response with the ALK inhibitor crizotinib. These results clearly show PF-06463922 to be a superior inhibitor of ALK kinase activity inhibiting all neuroblastoma mutant ALK forms assayed. Finally, single agent oral administration of PF-06463922 lead to induction of apoptosis and a dramatic reduction in tumour volume in a genetically engineered mouse model of treatment-resistant high-risk neuroblastoma driven by aberrant expression of MYCN and activated ALK. Taken together, our results suggest that PF-06463922 represents an important potential step forward in the treatment of relapsed neuroblastoma with mutated ALK. Statement of significance: Our results together with PK/PD analysis of PF-06463922 suggest future clinical trial investigation of ALK positive neuroblastoma Citation Format: J. Guan, L. Danielson, D. Chand, Y. Jamin, K. Ruuth, E. Tucker, G. Umapathy, A. El Wakil, B. Witek, T. W. Johnson, T. Smeal, L. Chesler, R. H. Palmer, B. Hallberg. The ALK inhibitor PF-06463922 shows significant response as a single agent in ALK/MYCN driven models of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B12.

B7-h3-specific engager T cells for the immunotherapy of pediatric solid tumors

Meeting abstracts B7-H3 positive tumors, including osteosarcoma, neuroblastoma, and high grade glioma, cause significant morbidity and mortality despite aggressive multimodality treatments. Current B7-H3-targeted immune-therapies take advantage of the monoclonal antibody 8H9, which is actively