Clinically aggressive pediatric spinal ependymoma with novel MYC amplification demonstrates molecular and histopathologic similarity to newly described MYCN-amplified spinal ependymomas

Primary spinal cord tumors contribute to ≤ 10% of central nervous system tumors in individuals of pediatric or adolescent age. Among intramedullary tumors, spinal ependymomas make up ~ 30% of this rare tumor population. A twelve-year-old male presented with an intradural, extramedullary mass occupying the dorsal spinal canal from C6 through T2. Gross total resection and histopathology revealed a World Health Organization (WHO) grade 2 ependymoma. He recurred eleven months later with extension from C2 through T1-T2. Subtotal resection was achieved followed by focal proton beam irradiation and chemotherapy. Histopathology was consistent with WHO grade 3 ependymoma. Molecular profiling of the primary and recurrent tumors revealed a novel amplification of the MYC (8q24) gene, which was confirmed by fluorescence in situ hybridization studies. Although MYC amplification in spinal ependymoma is exceedingly rare, a newly described classification of spinal ependymoma harboring MYCN (2p24) amplification (SP-MYCN) has been defined by DNA methylation-array based profiling. These individuals typically present with a malignant progression and dismal outcomes, contrary to the universally excellent survival outcomes seen in other spinal ependymomas. DNA methylation array-based classification confidently classified this tumor as SP-MYCN ependymoma. Notably, among the cohort of 52 tumors comprising the SP-MYCN methylation class, none harbor MYC amplification, highlighting the rarity of this genomic amplification in spinal ependymoma. A literature review comparing our individual to reported SP-MYCN tumors (n = 26) revealed similarities in clinical, histopathologic, and molecular features. Thus, we provide evidence from a single case to support the inclusion of MYC amplified spinal ependymoma within the molecular subgroup of SP-MYCN.

CSIG-08. ABT-414 RESISTANT GBM TUMORS HARBOR THE TEK S466I MUTATION AND EXHIBIT INCREASED STEMNESS

Glioblastoma (GBM), the most common primary brain tumor in adults, remains uniformly fatal due to the lack of effective targeted therapies for this aggressive malignancy. Genomic amplification of epidermal growth factor receptor (EGFR) occurs in 40-60% of primary GBM. Half of all EGFR-amplified cases of GBM also harbor EGFRvIII, a constitutively active, truncated variant of EGFR. The incidence of EGFR alterations in GBM makes inhibition of EGFR/EGFRvIII an attractive therapeutic approach. Depatuxizumab mafodotin (ABT-414) is an antibody-drug conjugate comprised of ABT-806, a mAB against EGFR, and a cytotoxic payload (monomethyl auristatin F). ABT-414 therapy initially showed promising results as an EGFRvIII therapy in vivo and in vitro; however, ABT414 failed to provide a survival benefit in phase I/II clinical trials, potentially due to therapeutic resistance. In this study, we seek to investigate the mechanisms of resistance to ABT-414 by performing whole exome, transcriptome and single cell RNA seq on ABT-414 resistant GBM PDX tumors. Our data showed an enrichment of mutations unique to the ABT-414 resistant tumors, including a point mutation (S466I) in TEK/TIE2 transmembrane angiopoietin receptor. In vitro expression of TEK S466I in GBM cells showed an increase in activation of ERK and STAT3 and increased TEK/TIE2 immunoprecipitation with EGFR compared to the wild-type TEK receptor. Furthermore, gene ontology analysis reveals that ABT-414-treated flank tumors exhibit increased activation of extracellular matrix organization and CNS developmental processes compared to flank tumors in the control treatment groups. ABT-414-treated tumors also demonstrate increased expression of inhibitor of differentiation (ID)1 and ID3, associated with stem-like phenotype in glioblastoma. Taken together, our data indicate that resistance to ABT-414 is mediated by both de novo mutations not detected in the parent tumor and adaptive dysregulation of pathways which may lead to dedifferentiation and therapeutic resistance.

Isolation and genomic amplification of fish and shrimps from mangrove ecosystems in North Sumatra

Mangrove is a very typical ecosystem with a high level of diversity of flora and fauna. However, mangrove ecosystems are also one of the most vulnerable locations to extinction due to the high disturbances that occur. Some of the species that are susceptible to such disturbances are fish and shrimp. The use of barcode DNA is considered a fairly effective way to evaluate the condition of fish and shrimp populations in North Sumatra. Therefore, this study aims to obtain information on the success of kit methods and amplification patterns of barcoding marks 16S for fish and shrimp in mangrove ecosystems North Sumatra. The results showed that the kit extraction method produced a pattern of band that varied thick, thin and even smear. PCR amplification results with the 16S gene showed a good band pattern and indicated that the 16S primer used can detect fish and shrimp species that are visualized in the form of DNA bands.

Whole genome sequencing of two human rhinovirus A types (A101 and A15) detected in Kenya, 2016-2018

Background: Virus genome sequencing is increasingly utilized in epidemiological surveillance. Genomic data allows comprehensive evaluation of underlying viral diversity and epidemiology to inform control. For human rhinovirus (HRV), genomic amplification and sequencing is challenging due to numerous types, high genetic diversity and inadequate reference sequences. Methods: We developed a tiled amplicon type-specific protocol for genome amplification and sequencing on the Illumina MiSeq platform of two HRV types, A15 and A101. We then assessed added value in analyzing whole genomes relative to the VP4/2 region only in the investigation of HRV molecular epidemiology within the community in Kilifi, coastal Kenya. Results: We processed 73 nasopharyngeal swabs collected between 2016-2018, and 48 yielded at least 70% HRV genome coverage. These included all A101 samples (n=10) and 38 (60.3%) A15 samples. Phylogenetic analysis revealed that the Kilifi A101 sequences interspersed with global A101 genomes available in GenBank collected between 1999-2016. On the other hand, our A15 sequences formed a monophyletic group separate from the global genomes collected in 2008 and 2019. An improved phylogenetic resolution was observed with the genome phylogenies compared to the VP4/2 phylogenies. Conclusions: We present a type-specific full genome sequencing approach for obtaining HRV genomic data and characterizing infections.

Multi-omics integration of methyltransferase-like protein family reveals clinical outcomes and functional signatures in human cancer

Human methyltransferase-like (METTL) proteins transfer methyl groups to nucleic acids, proteins, lipids, and other small molecules, subsequently playing important roles in various cellular processes. In this study, we performed integrated genomic, transcriptomic, proteomic, and clinicopathological analyses of 34 METTLs in a large cohort of primary tumor and cell line data. We identified a subset of METTL genes, notably METTL1, METTL7B, and NTMT1, with high frequencies of genomic amplification and/or up-regulation at both the mRNA and protein levels in a spectrum of human cancers. Higher METTL1 expression was associated with high-grade tumors and poor disease prognosis. Loss-of-function analysis in tumor cell lines indicated the biological importance of METTL1, an m7G methyltransferase, in cancer cell growth and survival. Furthermore, functional annotation and pathway analysis of METTL1-associated proteins revealed that, in addition to the METTL1 cofactor WDR4, RNA regulators and DNA packaging complexes may be functionally interconnected with METTL1 in human cancer. Finally, we generated a crystal structure model of the METTL1–WDR4 heterodimeric complex that might aid in understanding the key functional residues. Our results provide new information for further functional study of some METTL alterations in human cancer and might lead to the development of small inhibitors that target cancer-promoting METTLs.

Whole genome sequencing of two human rhinovirus A types (A101 and A15) detected in Kenya, 2016-2018

Background: Virus genome sequencing is increasingly utilized in epidemiological surveillance. Genomic data allows comprehensive evaluation of underlying viral diversity and epidemiology to inform control. For human rhinovirus (HRV), genomic amplification and sequencing is challenging due to numerous types, high genetic diversity and inadequate reference sequences. Methods: We developed a tiled amplicon type-specific protocol for genome amplification and sequencing on the Illumina MiSeq platform of two HRV types, A15 and A101. We then assessed added value in analyzing whole genomes relative to the VP4/2 region only in the investigation of HRV molecular epidemiology within the community in Kilifi, coastal Kenya. Results: We processed 73 samples collected between 2016-2018, and 48 yielded at least 70% HRV genome coverage. These included all A101 samples (n=10) and 38 (60.3%) A15 samples. Phylogenetic analysis revealed that the Kilifi A101 sequences interspersed with global A101 genomes available in GenBank collected between 1999-2016. On the other hand, our A15 sequences formed a monophyletic group separate from the global genomes collected in 2008 and 2019. Improved phylogenetic resolution was observed with the genome phylogenies compared to the VP4/2 phylogenies. Conclusions: We present a type-specific full genome sequencing approach for obtaining HRV genomic data and characterizing infections.

OTME-9. Comprehensive Metabolic Profiling Of high MYC Medulloblastoma Reveals Key Differences Between In Vitro And In Vivo Glucose And Glutamine Usage

Reprograming of cellular metabolism is a hallmark of cancer. The metabolic alterations in cancer cells is not only defined by series of genetic mutations, but also reflecting the crosstalk between cancer cells and other factors in the microenvironment. Altering metabolism allows cancer cells to overcome unfavorable conditions, to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. However, the metabolism of high MYC amplified medulloblastoma subgroup remains underexplored. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in different environments – in vitro, in flank xenografts and in orthotopic xenografts. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and the high-MYC medulloblastoma cells in culture. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of nucleotide, hexosamine biosynthetic pathway (HBP), TCA cycle, and amino acid and glutathione pathways. There was significantly higher glucose up taking and usage in orthotopic xenograft tumor compared to flank xenograft and cells in culture. The data demonstrated that glucose was the main carbon source for the glutamate, glutamine and glutathione synthesis through the TCA cycle. The glutaminase ii pathway was the main pathway utilizing glutamine in MYC-amplified medulloblastoma in vivo. Glutathione was found as the most abundant upregulated metabolite. Glutamine derived glutathione was mainly synthesized through glutamine transaminase K (GTK) enzyme in vivo. In conclusion, we demonstrated that high MYC medulloblastoma adapt to different environments by altering its metabolic pathways despite carrying the same genetic mutations. Glutamine antagonists may have therapeutic applications in human patients.

The pan-cancer lncRNA PLANE regulates an alternative splicing program to promote cancer pathogenesis

Genomic amplification of the distal portion of chromosome 3q, which encodes a number of oncogenic proteins, is one of the most frequent chromosomal abnormalities in malignancy. Here we functionally characterise a non-protein product of the 3q region, the long noncoding RNA (lncRNA) PLANE, which is upregulated in diverse cancer types through copy number gain as well as E2F1-mediated transcriptional activation. PLANE forms an RNA-RNA duplex with the nuclear receptor co-repressor 2 (NCOR2) pre-mRNA at intron 45, binds to heterogeneous ribonucleoprotein M (hnRNPM) and facilitates the association of hnRNPM with the intron, thus leading to repression of the alternative splicing (AS) event generating NCOR2-202, a major protein-coding NCOR2 AS variant. This is, at least in part, responsible for PLANE-mediated promotion of cancer cell proliferation and tumorigenicity. These results uncover the function and regulation of PLANE and suggest that PLANE may constitute a therapeutic target in the pan-cancer context.

Frequency and Prognostic Impact of ALK Amplifications and Mutations in the European Neuroblastoma Study Group (SIOPEN) High-Risk Neuroblastoma Trial (HR-NBL1)

PURPOSE In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS Genomic ALK amplification ( ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no- ALKa [n = 860] 51% [95% CI, 47 to 54], [ P < .001]), particularly in cases with metastatic disease. ALK mutations ( ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA ( P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively, P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

Molecular characterization of amplification of 11q13 region in patients with operable esophageal squamous cell carcinoma.

e16053 Background: The efficacy of programmed cell death 1 (PD-1) targeted therapy had showed effective treatment in patients (pts) with locally advanced or metastatic esophageal squamous cell carcinoma (ESCC). Meanwhile some studies have shown that PD-1 as neoadjuvant therapy showed promising anti-tumor activity with acceptable tolerance for locally advanced ESCC. Previous studies have shown genomic amplification of 11q13 region may serve as a negative predictive marker for ESCC patients receiving anti-PD-1 based immunotherapy. However, related molecular characterization study of amplification of 11q13 region in patients with operable ESCC is rare reported. Methods: The study recruited pts with operable ESCC whose tissues were send to perform target-capture deep sequencing with the pan-cancer panel and immunohistochemistry of PD-L1 with 22C3 assay. Pair-wise comparison of TMB and PD-L1 was analyzed in pts with 11q13 amplification and pts without 11q13 amplification. Results: 58 operable ESCC pts (cT1-T3N0) were enrolled, median age at diagnosis was 63 years(yrs) (45-73). Copy number analysis identified 30 out of 58 (52%) patients with amplifications of four genes located in chromosome 11q13 region, including CCND1(Cyclin D1) and fibroblast growth factor family members ( FGF3/4/19). Among the 11q13 amplification cohort, four genes amplified together were occurred in 27(90%) pts, 1(3%) patient had amplification of three genes ( CCND1 and FGF3/4), 2 (7%) pts had amplification of one gene that are FGF3 and CCND1 respectively. The prevalence rate of 11q13 amplification was not significantly different between older age (≥ 63 yrs) and young age ( < 63 yrs). Among 58 pts with TMB evaluated, median TMB was 5.76 muts/Mb (range: 0 - 20.16 muts/Mb). The prevalence of TMB-H and microsatellite instability was 12% (7/58) and 0% (0/58). TMB and expression of PD-L1 showed no significantly difference between 11q13 amplification cohort and 11q13 wild cohort. The most frequently mutated genes were TP53 (96.7%, 29/30 pts), NOTCH1 (33.3%, 10/30 pts), CDKN2A (23.3%, 7/30 pts) and MLL2 (20%, 6/30 pts) in 11q13 amplification cohort, while TP53 (89.3%, 25/28), NOTCH1 (39.3%,11/28), MLL2 (28.6%, 8/28), FAT1 (25%,7/28) were the most frequently mutated genes in 11q13 wild cohort. Conclusions: Amplification of 11q13 region was a high prevalence event in operable ESCC and there was show no age bias. Molecular characterization of amplification of 11q13 region in patients receiving PD-1 as neoadjuvant therapy before surgery may be meaningful.