Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such ...discoveries, the Children’s Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas and TP53 loss as a significant marker for poor overall survival in ependymomas and H3 K28-mutant diffuse midline gliomas. Already being actively applied to other pediatric cancers and PNOC molecular tumor board decision-making, OpenPBTA is an invaluable resource to the pediatric oncology community.
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•OpenPBTA collaborative analyses establish resource for 1,074 pediatric brain tumors•NGS-based WHO-aligned integrated diagnoses generated for 644 of 1,074 tumors•RNA-Seq analysis infers medulloblastoma subtypes, TP53 status, and telomerase activity•OpenPBTA will accelerate therapeutic translation of genomic insights
The OpenPBTA is a global, collaborative open-science initiative that brought together researchers and clinicians to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Shapiro et al. create over 40 open-source, scalable modules to perform cancer genomics analyses and provide a richly annotated somatic dataset across 58 brain tumor histologies. The OpenPBTA framework can be used as a model for large-scale data integration to inform basic research, therapeutic target identification, and clinical translation.
The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe ...cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system.
In order to dissect COVID-19-driven immune host responses, we performed RNA-seq of whole blood cell transcriptomes and granulocyte preparations from mild and severe COVID-19 patients and analyzed the data using a combination of conventional and data-driven co-expression analysis. Additionally, publicly available data was used to show the distinction from COVID-19 to other diseases. Reverse drug target prediction was used to identify known or novel drug candidates based on finding from data-driven findings.
Here, we profiled whole blood transcriptomes of 39 COVID-19 patients and 10 control donors enabling a data-driven stratification based on molecular phenotype. Neutrophil activation-associated signatures were prominently enriched in severe patient groups, which was corroborated in whole blood transcriptomes from an independent second cohort of 30 as well as in granulocyte samples from a third cohort of 16 COVID-19 patients (44 samples). Comparison of COVID-19 blood transcriptomes with those of a collection of over 3100 samples derived from 12 different viral infections, inflammatory diseases, and independent control samples revealed highly specific transcriptome signatures for COVID-19. Further, stratified transcriptomes predicted patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host.
Our study provides novel insights in the distinct molecular subgroups or phenotypes that are not simply explained by clinical parameters. We show that whole blood transcriptomes are extremely informative for COVID-19 since they capture granulocytes which are major drivers of disease severity.
•Analysis of deletion/insertion polymorphisms (DIPs) by digital droplet (dd) PCR represents a powerful technique for the sensitive and quantitative detection of overall and lineage-specific ...chimerism.•The DIP marker panel used was demonstrated to be informative in any donor-recipient constellation, including the related transplant setting.•Analysis of specific cell subsets isolated by flow sorting had a detection limit of .1%.•In terms of quantitative analysis, the results of ddPCR analysis were highly comparable to those of short tandem repeat PCR, which has been considered the gold standard in post-transplantation diagnostic testing of chimerism.
Analysis of specific leukocyte subsets for post-transplantation monitoring of chimerism provides greater sensitivity and clinical informativeness on dynamic changes in donor- and recipient-derived cells. Limitations of the most commonly used approach to chimerism testing relying on PCR-based analysis of microsatellite markers prompted us to assess the applicability of digital droplet (dd) PCR amplification of deletion/insertion polymorphisms (DIPs) for lineage-specific chimerism testing in the related stem cell transplantation setting, where the identification of informative markers facilitating the discrimination between donor-derived and recipient-derived cells can be challenging. We analyzed 100 genetically related patient-donor pairs by ddPCR analysis using commercially available DIP kits including large sets of polymorphic markers. At least 1 informative marker was identified in all related pairs analyzed, and 2 or more discriminating markers were detected in the majority (82%) of instances. The achievable detection limit is dependent on the number of cells available for analysis and was as low as 0.1% in the presence of ≥20,000 leukocytes available for DNA extraction. Moreover, the reproducibility and accuracy of quantitative chimerism analysis compared favorably to highly optimized microsatellite assays. Thus, the use of ddPCR-based analysis of DIP markers is an attractive approach to lineage-specific monitoring of chimerism in any allogeneic transplantation setting.
Abstract
Background
Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are ...not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing.
Methods
In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform.
Results
Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population.
Conclusions
Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.
Tracing of SARS-CoV-2 population transmission chains is still a major challenge. We present an integrated system of genomic surveillance and show it to be capable of detecting infection chains in a large city during ongoing community transmission.
Background: Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC, different oncolytic ...viruses (OVs) are currently investigated in clinical trials. However, systematic comparisons of these different OVs in terms of efficacy against PDAC and biomarkers predicting therapeutic response are lacking. Methods: We screened fourteen patient-derived PDAC cultures which reflect the intra- and intertumoural heterogeneity of PDAC for their sensitivity to five clinically relevant OVs, namely serotype 5 adenovirus Ad5-hTERT, herpes virus T-VEC, measles vaccine strain MV-NIS, reovirus jin-3, and protoparvovirus H-1PV. Live cell analysis, quantification of viral genome/gene expression, cell viability as well as cytotoxicity assays and titration of viral progeny were conducted. Transcriptome profiling was employed to identify potential predictive biomarkers for response to OV treatment. Findings: Patient-derived PDAC cultures showed individual response patterns to OV treatment. Twelve of fourteen cultures were responsive to at least one OV, with no single OV proving superior or inferior across all cultures. Known host factors for distinct viruses were retrieved as potential biomarkers. Compared to the classical molecular subtype, the quasi-mesenchymal or basal-like subtype of PDAC was found to be more sensitive to H-1PV, jin-3, and T-VEC. Generally, expression of viral entry receptors did not correlate with sensitivity to OV treatment, with one exception: Expression of Galectin-1 (LGALS1), a factor involved in H-1PV entry, positively correlated with H-1PV induced cell killing. Rather, cellular pathways controlling immunological, metabolic and proliferative signaling appeared to determine outcome. For instance, high baseline expression of interferon-stimulated genes (ISGs) correlated with relative resistance to oncolytic measles virus, whereas low cyclic GMP-AMP synthase (cGAS) expression was associated with exceptional response. Combination treatment of MV-NIS with a cGAS inhibitor improved tumour cell killing in several PDAC cultures and cells overexpressing cGAS were found to be less sensitive to MV oncolysis. Interpretation: Considering the heterogeneity of PDAC and the complexity of biological therapies such as OVs, no single biomarker can explain the spectrum of response patterns. For selection of a particular OV, PDAC molecular subtype, ISG expression as well as activation of distinct signaling and metabolic pathways should be considered. Combination therapies can overcome resistance in specific constellations. Overall, oncolytic virotherapy is a viable treatment option for PDAC, which warrants further development. This study highlights the need for personalised treatment in OVT. By providing all primary data, this study provides a rich source and guidance for ongoing developments. Funding: German National Science Foundation (Deutsche Forschungsgemeinschaft, DFG), German Cancer Aid (Deutsche Krebshilfe), German National Academic Scholarship Foundation (Studienstiftung des deutschen Volkes), Survival with Pancreatic Cancer Foundation.
Abstract 1408
About 10 % of patients with acute myeloid leukemia (AML) present with either monosomy 7 or deletions on the long arm of chromosome 7. This chromosomal aberration is associated with poor ...prognosis and adverse therapeutic response. Following biallelic inactivation as proposed by Knudsen’s hypothesis, a “second hit” of the remaining allele might be required for loss of gene function. So far, no consistent additional genetic hits could be identified in the minimally deleted regions. Thus, epigenetic silencing might display a local alternative mechanism ultimately causing silencing of a potential tumor suppressor. Based on cytogenetic data we defined a minimally deleted region on 7q22.2 of 2–3 Mb in size to narrow down the location of the putative tumor suppressor. It is flanked by the microsatellite markers D7S1503 and D7S1841. We utilized comprehensive DNA methylation profiling in the CpGrich areas of the minimal deleted regions by high resolution DNA methylation assessment by MassARRAY to identify aberrant epigenetic patterns in these regions. In this region we quantitatively analyzed CpG island methylation in a cohort of 64 AML patients with del(7q), 11 with monosomy 7, ten with normal karyotype and five CD34+ bone marrow cell DNA from healthy donors as controls. We identified four genes (PRES, LHFPL3, ATXN7L1, and CDH28) that are hypermethylated in their promoters in this region with significant mean methylation differences from 5 to 20 % comparing AML to healthy controls. Hypermethylation occurred both in patients with chromosome 7 aberrations and in patients with normal karyotypes. To narrow down aberrantly methylated genes to functional relevant candidates, we excluded those that were not expressed in healthy CD34+ cells. ATXN7L1, a gene located on 7q22.2 and coding for a potential subunit of the histone acetyl transferase (HAT) and deubiquitinase SAGA complex, was expressed in healthy CD34+ cells and granulocytes, but downregulated upon hypermethylation (>30%) in patients. To elucidate a potential functional role of ATXN7L1, we immunopurified SAGA from HeLa cells using antibodies against a SAGA subunit, GCN5,and analyzed the coimmunoprecipated proteins by subsequent mass spectrometry. Based on the identity of the co-precipitated proteins we demonstrate that ATXN7L1 is a bona fide component of the human SAGA complex. Thus our results suggest that ATXNL1 has a similar role as the closely related protein, ATXN7 that is known to be involved in transcription regulation via USP22 dependent deubiquitination of histones. Experiments to understand how ATXN7L1 silencing may affect the leukemic phenotype are in progress.
No relevant conflicts of interest to declare.
Abstract 3639
About 10 % of patients with either myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) present either with deletions on the long arm of chromosome 7 or monosomy 7. These ...chromosomal aberrations are associated with a poor prognosis. Following biallelic inactivation as proposed by Knudsen, a “second hit” of the remaining allele might be required for loss of gene function. Epigenetic silencing might display such a hit in particular since no additional genetic hits could be identified so far. The role of epigenetic regulation might be of special importance in patients with -7/7q-, since several studies have shown that patients harbouring a chromosome 7 abberrations do more benefit from a therapy with demethylating agents as compared to patients with other chromosomal changes.
To address the issue of epigenetic silencing in these AML cases we utilized DNA methylation profiling to identify the potential tumor suppressor genes on chromosome 7.
We used MBD2 based enrichment of methylated DNA from 4 AML patient samples with monosomy 7 and from 23 patient samples with with other chromosomal changes including normal karyotype AML. For validation we analyzed regional DNA methylation using quantitative MassArray technology on DNA from 115 del(7q) AML or monosomy 7 patients as well as normal karyotype patients (n=20) and CD34 + cells from healthy individuals (n=5).
We could identify the gene MEST to be silenced by hypermethylation (> 30 %) of a CpG island on the remaining allele in 20% of the patients with -7/7q- and 40% of patients with normal karyotype. Thus, preferentially in patients that do not have a deletion already.
MEST is an imprinted gene located on 7q32.2. However, silencing is correlated with hypermethylation of a CpG island located at an alternative promoter of MEST, independent of the methylation status of the imprinting control region.
In two patient samples with monosomy 7 and hypermethylation of MEST (> 80%), DNA methylation was erased after 5-Aza-2′-deoxycytidine (DAC) treatment to less than 10 %. In concordance, also AML cell lines with hypermethylation of the MEST locus loose methylation after sublethal DAC treatment which leads to a reexpression of the gene.
Thus, we hypothesize that MEST functions as a tumor suppressor in AML and is genetically as well as epigenetically silenced AML. Reactivation of MEST by demethylating treatment may contribute to the mechanism by which demethylating drugs display their therapeutic potential in leukemia.
No relevant conflicts of interest to declare.
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