Abstract 260: Application of integrated analysis of whole genome sequencing and RNA sequencing to personalized therapy decision making in pediatric and young adult cancer

Abstract Background: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder, occurs almost exclusively in smoking women and is characterized by a chronic polyclonal lymphocytosis with circulating binucleated lymphocytes, clonal cytogenetic abnormalities involving chromosome 3, and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies (non-Hodgkin´s lymphomas and solid tumors) were described. Potential molecular factors leading to their development are yet unclear. Aims: Detailed molecular genetic characterization of PPBL by whole genome sequencing (WGS) and RNA sequencing (RNAseq) in comparison to the well-characterized lymphoid malignancy CLL. Patient cohorts and methods: The total cohort comprised 27 PPBL (3 male, 24 female) and 250 CLL cases (163 male, 87 female). WGS was performed for all patients: 150bp paired-end reads where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies >1% where excluded. Final analysis was performed only on protein-altering and splice-site variants. For further analysis, a virtual panel of 355 lymphoid genes was selected. All reported p-values are two-sided and were considered significant at p<0.05. For gene expression analysis, estimated gene counts were normalized applying Trimmed mean of M-values (TMM) normalization method and the resulting log2 counts per million (CPMs) were used as a proxy of gene expression in each sample. Genes were kept if they were expressed (> 5 CPM) in at least 66% of the samples. Genes with FDR (false discovery rate) < 0.05 and an absolute logFC > 1.5 were considered differentially expressed (DE). Results: Median age was 46 years for PPBL patients (range: 23-67 years) and 67 years for CLL patients (range: 39-94 years). Mean number of mutations per patient was 18 for PPBL and 20 for CLL. For both entities, the majority of mutations were missense mutations (88% in PPBL vs. 81% in CLL), followed by splice-site mutations (7% vs. 10%), other mutation types were only rarely detected. In PPBL, 42 genes were found to be mutated at a frequency of >15%, including ATM (22%), CREBBP (19%), NCOR2 (19%), AHNAK2 (15%), JAK3 (15%), NOTCH2 (15%) and TRAF1 (15%), all of which have been associated with a variety of cancers. Moreover, ATM, NOTCH2 and TRAF1 mutations were described before to be associated with lymphomas. In PPBL patients, mutations in TRAF1 and ATM as well as mutations in TRAF1 and NOTCH2 were found to be mutually exclusive. For CLL patients, 29 genes showed a mutation frequency of >15%, comprising ATM (26%), KMT2D (23%), NOTCH1 (23%), LRP1B (19%), TP53 (16%) and CREBBP (15%). Comparison of the mutation frequencies between the two entities revealed several genes with significant differences: whereas mutations in CKAP5 (11% vs. 2%, p=0.022), DNMT3A (11% vs. 3%, p=0.033), MAP2 (19% vs. 4%, p=0.009), ROBO1 (15% vs. 4%, p=0.046) and TRAF1 (15% vs. 2%, p=0.006) were found to be more frequent in PPBL cases compared to CLL cases, KMT2D (4% vs. 23%, p=0.014), TDRD6 (0% vs. 14%, p=0.032) and TP53 (4% vs. 16%, p=0.048) mutations were more abundantly detected in CLL patients. Moreover, NOTCH1 was mutated more frequently in CLL cases (7% vs. 23%, p=0.082), whereas mutated NOTCH2 (known to be frequently mutated in splenic marginal zone lymphoma), was more abundant in PPBL patients (15% vs. 6%, p=0.116), although both correlations were not statistically significant. Gene expression analyses by RNAseq revealed 337 genes to be differentially expressed between the entities. 207 genes were upregulated in PPBL, including PTPRK, CXCR1, BCL11B, CEPBA, CCR4 and MYC, whereas 130 genes were found to be upregulated in CLL cases, comprising ID3, BCL2, FGF2 and FLT1. Conclusions: 1) WGS analysis identifies high frequencies of cancer/lymphoma-associated gene mutations in PPBL, including mutated ATM, NOTCH2 and TRAF1. 2) Five genes showed a higher mutation frequency compared to CLL including TRAF1,DNMT3A, CKAP5 and MAP2. 3) Lymphoma associated genes (BCL11B and MYC) were overexpressed in PPBL vs CLL. 4) Taken together our results question PPBL as a benign entity and identify molecular markers that might contribute to development of subsequent malignancies. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

Neurology Genetics ◽

10.1212/nxg.0000000000000554 ◽

2021 ◽

Vol 7 (1) ◽

pp. e554

Author(s):

Leigh B. Waddell ◽

Samantha J. Bryen ◽

Beryl B. Cummings ◽

Adam Bournazos ◽

Frances J. Evesson ◽

...

Keyword(s):

Creatine Kinase ◽

Western Blot ◽

Whole Genome Sequencing ◽

Rna Sequencing ◽

Genome Sequencing ◽

Premature Stop Codon ◽

Clinical Severity ◽

Whole Genome ◽

Wild Type ◽

Quantitative Western Blot

ObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.MethodsExome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.ConclusionsWhole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

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Whole-Genome and Transcriptome Sequencing Identified NOTCH2 and HES1 As Potential Markers of Response to Imatinib in Desmoid Tumor (Aggressive Fibromatosis): A Phase II Trial Study

10.21203/rs.3.rs-993539/v1 ◽

2021 ◽

Author(s):

Joonha Kwon ◽

Jun Hyeong Lee ◽

Young Han Lee ◽

Jeeyun Lee ◽

Jin-Hee Ahn ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Clinical Response ◽

Rna Sequencing ◽

Genome Sequencing ◽

Desmoid Tumor ◽

Large Scale ◽

Genetic Interaction ◽

Statistical Significance ◽

Aggressive Fibromatosis ◽

Whole Genome

Abstract Background: Desmoid tumor, also known as aggressive fibromatosis, is well-characterized by abnormal Wnt/β-catenin signaling. Various therapeutic options, including imatinib, are available to efficiently treat desmoid tumor. However, molecular mechanism of why imatinib works remains poorly understood. Here, we describe the potential roles of NOTCH2 and HES1 in association with clinical response to imatinib as in genome and transcriptome levels. Methods: We identified all somatic mutations in coding and non-coding regions via whole genome sequencing using desmoid tumor samples. To validate the genetic interaction with expression level in desmoid-tumor condition, we utilized large-scale Whole-genome sequencing (WGS) and transcriptome datasets from the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. RNA-sequencing was performed using prospective and retrospective cohort samples to evaluate the expressional relevance with clinical response. Results: Among 20 patients, 4 (20%) had a partial response and 14 (66.7%) had stable disease, 11 of which continued for ≥1 year. With gene-wise functional analyses, we detected significant correlation between recurrent NOTCH2 noncoding mutations and clinical response to imatinib. Based on PCAWG data analyses, NOTCH2 mutations affect its expression levels particularly in the presence of CTNNB1 missense mutations. By analyzing RNA-sequencing with additional desmoid tumor samples, we found that NOTCH2 expression was significantly correlated with HES1 expression. Interestingly, NOTCH2 had no statistical power to discriminate responders and non-responders. Instead, HES1 was differentially expressed with statistical significance between responders and non-responders Conclusions: Imatinib was effective and well tolerated for advanced desmoid tumor treatment. Our results show that HES1, regulated by NOTCH2, as an indicator of sensitivity to imatinib, which provides an important therapeutic consideration for desmoid tumor. Trial Registration: ClinicalTrials.gov, NCT02495519, Registered July 13, 2015- Retrospectively registered, https://www.clinicaltrials.gov/ct2/show/NCT02495519

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Integrated analysis of optical mapping and whole-genome sequencing reveals intratumoral genetic heterogeneity in metastatic lung squamous cell carcinoma

Translational Lung Cancer Research ◽

10.21037/tlcr-19-401 ◽

2020 ◽

Vol 9 (3) ◽

pp. 670-681

Author(s):

Yizhou Peng ◽

Chongze Yuan ◽

Xiaoting Tao ◽

Yue Zhao ◽

Xingxin Yao ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Squamous Cell ◽

Genetic Heterogeneity ◽

Lung Squamous Cell Carcinoma ◽

Integrated Analysis ◽

Whole Genome ◽

Metastatic Lung

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Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Viruses ◽

10.3390/v12080841 ◽

2020 ◽

Vol 12 (8) ◽

pp. 841 ◽

Cited By ~ 1

Author(s):

Carole Grädel ◽

Miguel A. Terrazos Miani ◽

Christian Baumann ◽

Maria Teresa Barbani ◽

Stefan Neuenschwander ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Rna Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

High Viral Load ◽

Clinical Samples ◽

Whole Genome ◽

Consensus Sequences ◽

Positive Stool ◽

Stool Samples

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

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Comprehensive Mutational Analysis of Juvenile Myelomonocytic Leukemia Using Whole-Genome Sequencing

Blood ◽

10.1182/blood-2019-121681 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2974-2974

Author(s):

Yusuke Okuno ◽

Hideki Muramatsu ◽

Norihiro Murakami ◽

Nozomu Kawashima ◽

Manabu Wakamatsu ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Rna Sequencing ◽

Genome Sequencing ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Driver Mutations ◽

Juvenile Myelomonocytic Leukemia ◽

Whole Genome ◽

Ras Pathway ◽

Myelomonocytic Leukemia

Background Juvenile myelomonocytic leukemia (JMML) is a rare and exclusively pediatric myelodysplastic/myeloproliferative neoplasm. This disease is genetically characterized by an extremely small number of somatic mutations (an average of 0.8 mutations/exome/patient). It has been shown that causative somatic and/or germline mutations activating the RAS pathway are located in PTPN11, NF1, NRAS, KRAS, and CBL in 85% of patients with JMML. Furthermore, up to 20% of the patients have additional secondary mutations including SETBP1, and JAK3 mutations. In 2% of the patients, we identified, by RNA sequencing, activating kinase lesions affecting ALK or ROS1. Such findings suggest that other kinase fusions are present in JMML. There is an exceptional scarcity of somatic passenger mutations on the exome, suggesting that a small number of driver mutations drive JMML. However, to date, this hypothesis has not been investigated by whole-genome sequencing. Patients and Methods We performed a whole-genome sequencing (WGS) study in 48 patients with JMML. Bone marrow specimens and in vitro-cultured T cells were used as tumor and germline samples, respectively. Next-generation sequencing was performed using a HiSeq X platform (Illumina). Data analysis was performed by our in-house pipeline. Specifically, the pipeline detects single nucleotide variants (SNVs), copy number variants, somatic loss of heterozygosity (LOH), and chromosomal structural variations (SVs). The study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results In each patient we detected an average of 28 somatic mutations. These were primarily C-to-T transition in the CpG context, indicating that the mutations occurred by cell division. Besides RAS pathway and known secondary mutations, we observed no significant accumulation of somatic mutations in either coding or non-coding regions. Although we detected RAS pathway mutations in 90% of the patients, all mutations were on exome. However, we identified germline microdeletions affecting CBL and NF1, which had not been identified by exome sequencing. Additionally, we found two LOH events that affected NF1. Bi-allelic inactivation of NF1 is generally observed in patients with JMML; however, no pathogenic SNVs were identified in these two patients. We identified two chromosomal translocations that caused activating kinase lesions (i.e., RANBP2-ALK and TBL1XR1-ROS1). These had been pointed out in our previous RNA sequencing study. Another patient carried a complex SV that affected XPO1 (encoding exportin 1 or chromosome region maintenance 1 protein homolog). Although fusion genes involving XPO1 are reported to be present in lymphoid malignancies, the role of this SV in JMML remains unclear. Conclusions JMML is characterized by driver mutations that are largely present within the exome. However, WGS can still play a role in identifying both coding and non-coding mutations. LOH events without pathogenic SNVs suggest the presence of novel regulatory mechanisms of NF1. Conclusively, JMML is characterized by a paucity of somatic alterations and driver mutations. Hence, current research efforts should focus on RAS pathway mutations and known secondary mutations, many of which can be targeted. Disclosures No relevant conflicts of interest to declare.

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Impact of cobas PCR Media Freezing on SARS-CoV-2 Viral RNA Integrity and Whole Genome Sequencing Analyses

10.1101/2021.02.05.430022 ◽

2021 ◽

Author(s):

Patrick Benoit ◽

Floriane Point ◽

Simon Gagnon ◽

Daniel E. Kaufmann ◽

Cécile Tremblay ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Rna Sequencing ◽

Genome Sequencing ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Freezing And Thawing ◽

Rna Integrity ◽

Transport Medium

ABSTRACTSARS-CoV-2 whole genome sequencing is an important molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swab samples and shipment to reference laboratories is usually required since RNA sequencing is rarely available in routine clinical microbiology laboratories where initial diagnosis and support to outbreak investigations occur. The cobas PCR Media transport medium developed by Roche facilitates high throughput analyses on cobas multianalyzer PCR platforms. There is no data on the stability of SARS-CoV-2 RNA after freezing and thawing of clinical samples in this transport medium, but potential denaturing of the molecular template could impair test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral whole genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Genomic coverage and sequencing depth did not significantly differ between fresh and frozen samples (n=10). For samples with lower viral inoculum and PCR cycle threshold above 30, sequencing quality scores and detection of single nucleotide polymorphisms did not differ either. Freezing of cobas PCR Media does not negatively affect the quality of SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories. Those results support secondary use of diagnostic nasopharyngeal swab material for viral sequencing without requirement for additional clinical samples.

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