scholarly journals HGG-09. A ROBUST CELL-FREE DNA (CFDNA) ASSAY TO DETECT MUTATIONS IN PLASMA SAMPLES OF PEDIATRIC HIGH GRADE GLIOMA PATIENTS DESPITE GENOMIC DNA (GDNA) CONTAMINATION

2019 ◽  
Vol 21 (Supplement_2) ◽  
pp. ii88-ii88
Author(s):  
Agda Karina Eterovic ◽  
Nader Ezzeddine ◽  
Ping Song ◽  
Lin-ya Tang ◽  
Mohammad Mohammad ◽  
...  
Keyword(s):  
Genomic Dna ◽  
High Grade Glioma ◽  
High Grade ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Free Dna ◽  
Gdna Contamination ◽  
Pediatric High Grade Glioma

scholarly journals Synthetic Circulating Cell-free DNA as Quality Control Materials for Somatic Mutation Detection in Liquid Biopsy for Cancer

2017 ◽  
Vol 63 (9) ◽  
pp. 1465-1475 ◽  
Author(s):  
Rui Zhang ◽  
Rongxue Peng ◽  
Ziyang Li ◽  
Peng Gao ◽  
Shiyu Jia ◽  
...  
Keyword(s):  
Quality Control ◽  
Somatic Mutation ◽  
Genomic Dna ◽  
Mutation Detection ◽  
Micrococcal Nuclease ◽  
Plasma Samples ◽  
Cell Free Dna ◽  
Free Dna ◽  
Quality Control Materials ◽  
Somatic Mutation Detection

AbstractBACKGROUNDDetection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently.METHODSWe developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500–2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories.RESULTSThe results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFR T790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2.CONCLUSIONSThe SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.

scholarly journals BIOM-56. THE INTEGRATION OF A LIQUID BIOPSY PROGRAM INTO THE CARE OF PEDIATRIC BRAIN TUMOR PATIENTS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii13-ii14
Author(s):  
Alexandra Miller ◽  
Luca Szalontay ◽  
Hamza Ahmad ◽  
Nancy Bouvier ◽  
Irene Rodriguez-Sanchez ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Tumor Tissue ◽  
Clinical Care ◽  
High Grade Glioma ◽  
Response To Therapy ◽  
Low Grade ◽  
High Grade ◽  
Cell Free Dna ◽  
Free Dna ◽  
Minimal Residual

Abstract Pediatric CNS tumors remain the leading cause of cancer-related death in children and adolescents. Safe sampling of tumor tissue for diagnostic purposes may be difficult if not impossible. Detection of minimal residual or recurrent disease prior to definitive clinical or radiographic progression may allow earlier initiation of novel therapies and ultimately improve overall survival. Given the rarity of serial sampling of tumor tissue, our understanding of molecular evolution in response to therapy remains limited. Recent technological advances have led to the development of “liquid biopsy” assays, which detect cell-free DNA (cfDNA) in blood, cerebrospinal fluid (CSF) or other bodily fluids. Here, we report our initial clinical experience with the recently established MSK Kids pediatric neuro-oncology liquid-biopsy program at Memorial Sloan Kettering Cancer Center (MSKCC) using MSK-IMPACT, which is clinically validated by the New York State Department of Health for CSF cell-free DNA (cfDNA)vprofiling. All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. Samples from 29 unique patients were sequenced. Histopathology included high-grade glioma (5); low-grade glioma (2); medulloblastoma (8); pineoblastoma (3); retinoblastoma (4); other (7). CSF cfDNA could be detected in 18/42 samples (43%) and 12/29 patients (34%). CSF cfDNA was more commonly detected in higher-grade, disseminated tumors such as high-grade glioma (60%), medulloblastoma (38%), and pineoblastoma (100%). Low-grade lesions without leptomeningeal involvement did not result in detectable CSF cfDNA shedding (86% were negative). In a subset of patients, MSK-IMPACT identified previously unrecognized molecular actionable targets (e.g. BRAF-KIAA1549 fusion); or the detection of “minimal residual disease” prior to the detection of tumor recurrence by conventional diagnostics, impacting clinical care decisions. Future directions include integration of CSF cfDNA into prospective clinical trials as a correlative biomarker.

scholarly journals HG-10 * HYPOXIA PATHWAY DRIVEN BY HIF-1ALPHA ARE PREDOMINANTLY INVOLVED IN PEDIATRIC HIGH GRADE GLIOMA AND REPRESENT PROMISING THERAPEUTIC TARGETS

2015 ◽  
Vol 17 (suppl 3) ◽  
pp. iii12-iii12
Author(s):  
C. Lasthaus ◽  
M. Litzler ◽  
C. Bour ◽  
D. Guenot ◽  
N. Entz-Werle
Keyword(s):  
Therapeutic Targets ◽  
High Grade Glioma ◽  
High Grade ◽  
Pediatric High Grade Glioma

scholarly journals Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma

2018 ◽  
Vol 115 (46) ◽  
pp. E10925-E10933 ◽  
Author(s):  
Peiyong Jiang ◽  
Kun Sun ◽  
Yu K. Tong ◽  
Suk Hang Cheng ◽  
Timothy H. T. Cheng ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Circulation ◽  
Sequence Information ◽  
Sequencing Analysis ◽  
Plasma Samples ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Dna

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

scholarly journals Non-Invasive Prenatal Diagnosis (NIPD) of Sickle-Cell Disease By Massively Parallel Sequencing of Cell-Free Fetal DNA in Maternal Serum

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2085-2085
Author(s):  
Yvonne Daniel ◽  
Julia Van Campen ◽  
Lee Silcock ◽  
Michael Yau ◽  
Joo Wook Ahn ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Massively Parallel Sequencing ◽  
Plasma Samples ◽  
Cell Disease ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Cell Free Fetal Dna

Sickle cell disease (SCD) is the most common genetic haematological disorder worldwide. Around 300.000 affected infants are born every year, including at least 1000 in the United States. Prenatal diagnosis is currently carried out using amniotic fluid or chorionic villus sampling. These invasive procedures are perceived to have a small risk of miscarriage. The availability of non-invasive prenatal diagnosis (NIPD) is predicted to increase uptake of prenatal diagnosis for SCD, as it has no perceived miscarriage risk. NIPD may also be more readily implemented than invasive prenatal diagnosis in the low-resource countries in which SCD is the most prevalent. However, accurate NIPD of autosomal recessive disorders such as sickle cell disease has proven challenging as this requires detection of fetal inheritance of a maternal allele from a mixed maternal-fetal pool of cell-free DNA. We report the development of a targeted massively parallel sequencing assay for the NIPD of fetal SCD using cell-free fetal DNA from maternal plasma. No paternal or previous offspring samples were required. 44 clinical samples were analysed, including 37 plasma samples from pregnant SCD carriers and 7 plasma samples from women with SCD due to Hb SC. We used a relative mutation dosage based approach for the 37 samples from maternal SCD carriers (Hb AS or Hb AC), integrating Unique Molecular Identifiers (UMIs) into the analysis to improve the accuracy of wildtype and mutant allele counts. We used a separate wildtype allele detection approach for the 7 samples from women with compound heterozygous SCD, in whom the detection of wildtype cell-free DNA indicates the presence of a carrier fetus. The success of the assay was evaluated by comparing results with the established fetal sickle status as determined through either invasive prenatal diagnosis or newborn screening. During development, two key factors improved the accuracy of the results: i) Selective analysis of only smaller cell-free DNA fragments enhanced the fetal fraction for all samples, with greater effects observed in samples from earlier gestations. This approach improved diagnostic accuracy: for 3 out of 44 samples, the genotype was inconclusive or incorrect before size selection, but correct after size selection. ii) Modifications to DNA fragment hybridisation capture optimised the diversity of Unique Molecular Identifier-tagged molecules analysed. This led to improvements in the results obtained for 5 samples, with 3 previously inconclusive samples correctly called and 2 previously discrepant results moved into the inconclusive range. In total, 37 results were concordant with the established fetal sickle status; this included 30/37 samples from carrier women and 7/7 samples from women with sickle cell disease due to Hb SC. The remaining 7 carrier samples gave an inconclusive result, which for 3 samples was attributed to a low fetal fraction. Samples from as early as 8 weeks gestation were successfully genotyped. There were no false positive or false negative results. This study is the largest to use NGS-based NIPD on clinical plasma samples from pregnancies at risk of SCD. Efforts to validate the assay on a larger sample cohort and to reduce the inconclusive rate are warranted. This study shows that NIPD for SCD is approaching clinical utility and has the potential to provide increased choice to women with pregnancies at risk of sickle cell disease. Disclosures Silcock: Nonacus Ltd.: Employment.

scholarly journals HGG-41. STRUCTURAL VARIANT DRIVERS IN PEDIATRIC HIGH-GRADE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii351-iii351
Author(s):  
Frank Dubois ◽  
Ofer Shapira ◽  
Noah Greenwald ◽  
Travis Zack ◽  
Jessica W Tsai ◽  
...  
Keyword(s):  
Copy Number ◽  
High Grade Glioma ◽  
High Grade ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Effector Domains ◽  
Topologically Associating Domains ◽  
Genome Wide ◽  
Pediatric High Grade Glioma

Abstract BACKGROUND Driver single nucleotide variants (SNV) and somatic copy number aberrations (SCNA) of pediatric high-grade glioma (pHGGs), including Diffuse Midline Gliomas (DMGs) are characterized. However, structural variants (SVs) in pHGGs and the mechanisms through which they contribute to glioma formation have not been systematically analyzed genome-wide. METHODS Using SvABA for SVs as well as the latest pipelines for SCNAs and SNVs we analyzed whole-genome sequencing from 174 patients. This includes 60 previously unpublished samples, 43 of which are DMGs. Signature analysis allowed us to define pHGG groups with shared SV characteristics. Significantly recurring SV breakpoints and juxtapositions were identified with algorithms we recently developed and the findings were correlated with RNAseq and H3K27ac ChIPseq. RESULTS The SV characteristics in pHGG showed three groups defined by either complex, intermediate or simple signature activities. These associated with distinct combinations of known driver oncogenes. Our statistical analysis revealed recurring SVs in the topologically associating domains of MYCN, MYC, EGFR, PDGFRA & MET. These correlated with increased mRNA expression and amplification of H3K27ac peaks. Complex recurring amplifications showed characteristics of extrachromosomal amplicons and were enriched in coding SVs splitting protein regulatory from effector domains. Integrative analysis of all SCNAs, SNVs & SVs revealed patterns of characteristic combinations between potential drivers and signatures. This included two distinct groups of H3K27M DMGs with either complex or simple signatures and different combinations of associated variants. CONCLUSION Recurrent SVs associate with signatures shaped by an underlying process, which can lead to distinct mechanisms to activate the same oncogene.

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