Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model.

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.

Added Value of Reanalysis of Whole Exome- and Whole Genome Sequencing Data From Patients Suspected of Primary Immune Deficiency Using an Extended Gene Panel and Structural Variation Calling.

Background: Knowledge of the genetic variation underlying Primary Immune Deficiency (PID) is increasing. Reanalysis of genome-wide sequencing data from undiagnosed patients with suspected PID may improve the diagnostic rate. Methods: We included patients monitored at the Department of Infectious Diseases or the Child and Adolescent Department, Rigshospitalet, Denmark, for a suspected PID, who had been analysed previously using a targeted PID gene panel (457 PID-related genes) on whole exome- (WES) or whole genome sequencing (WGS) data. A literature review was performed to extend the PID gene panel used for reanalysis of single nucleotide variation (SNV) and small indels. Structural variant (SV) calling was added on WGS data. Results: Genetic data from 94 patients (86 adults) including 36 WES and 58 WGS was reanalysed a median of 23 months after the initial analysis. The extended gene panel included 208 additional PID-related genes. Genetic reanalysis led to a small increase in the proportion of patients with new suspicious PID related variants of uncertain significance (VUS). The proportion of patients with a causal genetic diagnosis was constant. In total, five patients (5%, including three WES and two WGS) had a new suspicious PID VUS identified due to reanalysis. Among these, two patients had a variant added due to the expansion of the PID gene panel, and three patients had a variant reclassified to a VUS in a gene included in the initial PID gene panel. The total proportion of patients with PID related VUS, likely pathogenic, and pathogenic variants increased from 43 (46%) to 47 (50%), as one patient had a VUS detected in both initial- and reanalysis. In addition, we detected new suspicious SNVs and SVs of uncertain significance in PID candidate genes with unknown inheritance and/or as heterozygous variants in genes with autosomal recessive inheritance in 8 patients. Conclusion: These data indicate a possible diagnostic gain of reassessing WES/WGS data from patients with suspected PID. Reasons for the possible gain included improved knowledge of genotype-phenotype correlation, expanding the gene panel, and adding SV analyses. Future studies of genotype-phenotype correlations may provide additional knowledge on the impact of the new suspicious VUSs.

Whole-Genome Sequencing for Investigating a Health Care-Associated Outbreak of Carbapenem-Resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAB) outbreaks in hospital settings challenge the treatment of patients and infection control. Understanding the relatedness of clinical isolates is important in distinguishing outbreak isolates from sporadic cases. This study investigated 11 CRAB isolates from a hospital outbreak by whole-genome sequencing (WGS), utilizing various bioinformatics tools for outbreak analysis. The results of multilocus sequence typing (MLST), single nucleotide polymorphism (SNP) analysis, and phylogenetic tree analysis by WGS through web-based tools were compared, and repetitive element polymerase chain reaction (rep-PCR) typing was performed. Through the WGS of 11 A. baumannii isolates, three clonal lineages were identified from the outbreak. The coexistence of blaOXA-23, blaOXA-66, blaADC-25, and armA with additional aminoglycoside-inactivating enzymes, predicted to confer multidrug resistance, was identified in all isolates. The MLST Oxford scheme identified three types (ST191, ST369, and ST451), and, through whole-genome MLST and whole-genome SNP analyses, different clones were found to exist within the MLST types. wgSNP showed the highest discriminatory power with the lowest similarities among the isolates. Using the various bioinformatics tools for WGS, CRAB outbreak analysis was applicable and identified three discrete clusters differentiating the separate epidemiologic relationships among the isolates.

Investigation of a Carbapenemase-producing Acinetobacter baumannii outbreak using whole genome sequencing versus a standard epidemiologic investigation.

Background: The standard epidemiologic investigation of outbreaks typically relies on spatiotemporal data and pulsed-field gel electrophoresis (PFGE), but whole genome sequencing (WGS) is becoming increasingly used. This investigation aimed to characterize a carbapenemase-producing Acinetobacter baumannii (CPAb) nosocomial outbreak using WGS compared to a standard outbreak investigation. Methods: The CPAb outbreak occurred in a single center between 2012 and 2014. The standard investigation used spatiotemporal data and PFGE to generate a chain of transmission. A separate WGS investigation generated a chain of transmission based solely on WGS and date of sampling and was blinded to all other spatiotemporal data and PFGE. Core single nucleotide variant (SNV) phylogenetic analysis was performed on WGS data generated using the Illumina MiSeq platform. The chains of transmission were compared quantitatively and qualitatively to assess the concordance between both methods. Results: 28 colonized and infected cases were included. Of the 27 transmission events identified using the standard investigation, 12 (44%) were identical to the transmission events using WGS. WGS identified several transmission events that had not been detected by traditional method, and numerous transmission events that had occurred on different hospital wards than suspected by standard methods. The average number (standard deviation [SD]) of SNVs per transmission events was 1.63 (SD, 1.31) by traditional method and 0.63 (SD, 0.79) by WGS (p = 0.001) All isolates harbored the rare carbapenemase blaOXA-237. Conclusions: The traditional and WGS investigations had moderate concordance. When used alongside epidemiologic data and clinical information, WGS could help improve the mapping of transmission events.