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Item Genomic Landscape of Epstein-Barr Virus in Endemic Burkitt Lymphoma(Elsevier, 2023-11-29) Legason, Ismail Draguma; Burns, Adam; Ridout, Kate; Jennings, Daisy; Ogwang, Martin D; Otim, Isaac; Nabalende, Hadijah; Achola, Caroline; Ayitewala, Alisen; Sseruyange, Julius; Chamba, Clara C; Josephat, Emmanuel; Christopher, Heavenlight; Mawalla, William F; Mkwizu, Elifurah; Mremi, Alex; Mwamtemi, Hadija; Dreau, Helene; Mouden, Claire El; Vavoulis, Dimitris; Leung, Carol Sze; Schuh, AnnaIntroduction: Endemic Burkitt lymphoma (eBL) is a fast-growing B-cell malignancy strongly linked to Epstein-Barr virus (EBV) infection, a member of the herpes virus family. The virus is widespread, infecting 95% of the global population, and has also been linked to several other cancers, including nasopharyngeal carcinoma (NPC), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), natural killer T-cell lymphoma (NKTCL), gastric carcinoma (GC) and leiomyosarcomas. The distribution of these cancers is inconsistent in different regions, raising the hypothesis of whether different strains are linked to specific cancers. Using high throughput sequencing, we obtained EBV whole genome sequences from the plasma of children with eBL diagnosis from a large cohort study in East Africa. Methods: Twenty-one EBV-positive patients (Sixteen BL, one diffuse large B-cell lymphoma, one lymphoblastoid lymphoma, one clear cell sarcoma, one small blue cell tumour and one alveolar rhabdomyosarcoma) were recruited from St Mary's Hospital Lacor, in Uganda, between 2020 and 2023. Venous blood samples were collected in appropriate DNA tubes, and ~ 5 ml of plasma was used for cfDNA extraction using QIAamp circulating Nucleic acid kit (Qiagen, USA). The cfDNA libraries were prepared using the Thruplex Tag- Seq kit (Takara Bio Inc. Japan), target capture and enrichment was performed using an EBV-custom panel (IDT Inc. USA) before sequencing. The Illumina fastq files were processed using an in-house pipeline, and paired-end reads were aligned to a bespoke hybrid EBV reference genome (NC_007605.1 and EBNA genes from NC_009334) using BWA with an alt-ware method. The average sequencing depth and EBV genome coverage were 5164 and 97.8%, respectively. Following the GATK best practice workflows (version 4.0), 15,318 variants were called across all samples using Varscan and Vardict after base and variant recalibration. Functional annotation was performed using the SNPEff package (v5.1d) according to the reference genome (NC_007605.1, NCBI annotation, Nov 2013). For the phylogenetic analysis, 322 public EBV genomes were downloaded from the GenBank and aligned with 21 EBV genomes from the current study using a multiple sequence alignment program (MAFFT v7.52), and the maximum likelihood of the phylogenetic relationship was inferred using RAxML (v8.0), assuming a general time reversible (GTR) model. The inferred phylogeny was subsequently rooted using the evolutionary placement (EPA) algorithm from RAxML using a Macacine herpesvirus 4 genome sequence (NC_006146) as an outer group. The tree was annotated using FigTree software (v1.4.4). The study was approved by the Oxford Tropical Research Ethics Committee (OxTREC Ref: 15-19), National Institute of Medical Research (NIMR/HQ/R.8a/Vol.IX/3408), Uganda National Council of Science and Technology (Ref: HS529ES), and St Mary's Hospital Lacor Institutional Research Ethics Committee. Participants provided written informed consent and assent for minors. Results: We examined the genomes of 21 EBV samples, including 18 type-1 and 3 type-2 strains. Our analysis revealed an average of 729 mutations per sample, comprised of 707 SNVs and 26 indels (<50bp) per sample (Figure 1). Within 16 eBL tumours, we observed the 30-bp deletion in LMP-1 in 43.8% (7/16) cases. This deletion has been previously reported in NPC in Southeast Asia and is associated with high oncogenic potential and weak immunogenicity. We also identified a novel frameshift variant (15-bp deletion) in LMP-1 in 66.7% (14/21) of tumour samples. The LMP-1 gene encodes a 356-amino acid polypeptide with a cytoplasmic amino terminus, a transmembrane domain, and a cytoplasmic carboxy terminus. The C-terminus interacts with cellular proteins, activating the transcriptional nuclear factor-kB (NF-kB) pathway associated with antiapoptotic cell signalling. Additionally, we observed a high degree of polymorphism in EBNA-1, EBNA3s, LMP-2A, BALF1, and BOLF-1 viral genes. Our phylogenetic analysis revealed distinct clustering of African EBV genomes compared to Asian strains and NPC versus eBL (Figure 2). Conclusions: Overall, our study provides the first-ever comprehensive mutational profile of EBV in eBL clinical samples, offering valuable insights into the somatic events of EBV in tumour biology.Item Performance of a liquid biopsy diagnostic prediction model for EBV-positive Burkitt Lymphoma in Sub-Saharan Africa(Elsevier, 2023-11-29) Chamba, Clara C; Howard, Kieran; Mawalla, William F; Christopher, Heavenlight; Seruyange, Julius; Josephat, Emmanuel; Legason, Ismail Draguma; Ogwang, Martin; Ayitewala, Alisen; Mremi, Alex; Achola, Caroline; Elias, Edrick; Cutts, Anthony; Dreau, Helene; Burns, Adam; Shungu, Rehema; Gerlevik, Sila; Jennings, Daisy; Balandya, Emmanuel; Ridout, KateBurkitt Lymphoma (BL) is classified into EBV-positive BL (EBL) and EBV-negative BL (sporadic BL), the former being more prevalent in SSA (Al-Khreisat et al 2023). Conventional diagnosis of BL depends on the morphological assessment of invasive tissue biopsy followed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) to reveal the pathognomonic t(8;14), or t(8;22) translocations. This is associated with significant diagnostic delay in regions with limited pathology services (Mawalla et al 2023), often necessitating initiation of treatment based on clinical or histopathological findings without immunohistochemistry (Ogwang et al 2011), with ensuing mismanagement contributing to poor survival outcomes observed in SSA compared to High-Income Countries (HIC) (Sung et al 2021). Here, we sought to explore whether liquid biopsy could be used as an initial diagnostic aid for EBL in settings without easy access to pathology using different EBV DNA parameters and the MYC-Immunoglobulin ( MYC-Ig) gene rearrangement. This prospective study collected whole blood samples prospectively from 221 participants (90 BL and 131 non-BL) enrolled in the Aggressive Infection-Related East Africa Lymphoma (AI-REAL) project (Legason et al 2022) across 5 sites in Tanzania and Uganda. The revised BL diagnostic algorithm (Naresh et al 2021) was used as a gold-standard diagnosis. CfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, USA), according to the manufacturer's instructions. DNA libraries were prepared with the Thru PLEX Tag-Seq HV kit (Takara Bio Inc. Japan), and hybridization capture was performed using a custom, next-generation sequencing (NGS) panel targeting genes commonly mutated in BL and three EBV genes: EBER1, EBER2, and EBNA2. All sequencing was performed in-country (MiSeq, Illumina, CA, USA) and analyzed in a custom pipeline. Using custom scripts, EBV DNA copies per cell and EBV DNA fragment size ratio were calculated as the proportion of EBV fragments of length 180 - 200 base pairs (bp) divided by the proportion of autosomal fragments within the same size window. The diversity in the fragment size distribution of the EBV and autosomal DNA was calculated and presented as EBV entropy and autosomal entropy. Translocations were called using IgCaller (Nadeu et al 2020) and Genomic Rearrangement Identification Software Suite (GRIDSS) (Cameron et al 2017). Univariate analysis was performed to assess the association between predictor variables (clinical parameters, EBV parameters, and MYC translocation) and a diagnosis of EBL. Ten-fold cross-validation was performed on a liquid biopsy model consisting of all the liquid biopsy variables with the clinical variables included in the model as covariates. Performance metrics for the liquid biopsy model as a diagnostic aid were calculated, and variable importance was determined to assess each variable's relative influence on the model. In our cohort, EBL diagnosis was negatively associated with tumor site (neck) and not significantly associated with tumor site (jaw). A shorter duration of symptoms was associated with a diagnosis of EBL. Presence of MYC translocation t(8;14), higher EBV fragment size ratio, proportion, entropy, and copies per cell for EBER1, EBER2, and EBNA2 were all associated with a diagnosis of EBL.The combined liquid biopsy diagnostic prediction model had an estimated AUC of 90%, with sensitivity between 85%-90%, specificity between 75% - 80%, and positive and negative predictive values of 85% (Figure 1). MYC translocation t(8;14) was the most important diagnostic variable, followed by EBV entropy (Figure 2). This study demonstrates the feasibility of liquid biopsy as a non-invasive diagnostic aid for EBL in settings without easy access to pathology. To strengthen the application of this technology into clinical practice, further analysis is being conducted for weighting and assigning of scores for each predictor variable. This would assist clinicians in making a precise diagnosis, complementing existing diagnostic modalities for EBL, and reducing the rate of misdiagnosis arising from diagnosis based on clinical suspicion and tissue morphology without immunohistochemistry.