FrbJ is a member of the Fe(2+)/α-ketoglutarate-dependent dioxygenase family which hydroxylates the natural product FR-900098 of Streptomyces rubellomurinus, yielding the phosphonate antibiotic FR-33289. Here, the crystal structure of FrbJ, which shows structural homology to taurine dioxygenase (TauD), a key member of the same family, is reported. Unlike other members of the family, FrbJ has an unusual lid structure which consists of two β-strands with a long loop between them. To investigate the role of this lid motif, a molecular-dynamics simulation was performed with the FrbJ structure. The molecular-dynamics simulation analysis implies that the lid-loop region is highly flexible, which is consistent with the fact that FrbJ has a relatively broad spectrum of substrates with different lengths. Interestingly, an access tunnel is found at the back of the active site which connects the putative binding site of α-ketoglutarate to the solvent outside.

BackgroundThis study aimed to develop a vaccine that targets mutation-derived neoantigen in Chinese non-small-cell lung cancer (NSCLC).MethodsA cohort of 1862 Chinese NSCLC patients who underwent targeted sequencing with a 1021-gene panel was investigated. HLA typing was done using OptiType v1.0 and neoantigens were predicted by netMHCpan v4.0. HLA LOH was inferred using the lohhla algorithm and TMB were quantified by counting the total number of non-synonymous ones based on our panel data. CIBERSORT was utilized to estimate the TME in different EGFR mutant subtype by using TCGA data.ResultsHLA-A*11:01(42.59%) was the top one allele and HLA-A*33:03(12.94%) ranked 12th. EGFR L858R (22.61%) was the most prevalent gene variant. The binding affinity (IC50 MT = 22.9 nM) and shared frequency (2.93%) of EGFR L858R in combination with HLA-A*33:03 were optimal. In a subsequent further analysis on immunological features of EGFR mutant subtypes, 63.1% HLA loss of heterozygosity LOH (HLA LOH) and 0.37% (7 of 1862) B2M aberrations were found in our population, both had no significant association with EGFR mutant subtypes suggesting that the process of antigen presentation involved HLA LOH and B2M mechanisms in EGFR L858R is working. Tumor mutation burden (TMB) was investigated by utilizing our panel and showed that EGFR L858R had the lowest TMB compared with other EGFR mutant subtypes. In addition, analysis of 22 immune cell types from The Cancer Genome Atlas (TCGA) data showed EGFR L858R was correlated with low level of CD8 T cells, activated CD4 memory T cells and elevated level of macrophage M2 suggesting an inhibited tumor microenvironment (TME).ConclusionOur study identified that EGFR L858R neoantigen had the potential to generate cancer vaccines in NSCLC patients with HLA A*33:03. The neoantigen-based vaccines may become an effective salvage regimen for EGFR L858R subgroup after targeted therapy or immune checkpoint inhibitors (ICIs) failure.

Background Tumor mutational burden (TMB) is a promising biomarker for stratifying patient subpopulation who would benefit from immune checkpoint blockade (ICB) therapies. Although great efforts have been made for standardizing TMB measurement, mutation calling and TMB quantification can be challenging in samples with low tumor content including liquid biopsies. The effect of varying tumor content on TMB estimation by different assay methods has never been systematically investigated. Method We established a series of reference standard DNA samples derived from 11 pairs of tumor–normal matched human cell lines across different cancer types. Each tumor cell line was mixed with its matched normal at 0% (control), 1%, 2%, 5%, and 10% mass‐to‐mass ratio to mimic the clinical samples with low tumor content. TMB of these reference standards was evaluated by both ∼1000× whole‐exome sequencing (wesTMB) and targeted panel sequencing (psTMB) at four different vendors. Both regression and classification analyses of TMB were performed for theoretical investigation and clinical practice purposes. Results Linear regression model was established that demonstrated in silico psTMB determined by regions of interest (ROI) as a great representative of wesTMB based on TCGA dataset. It was also true in our reference standard samples as the predicted psTMB interval based on the observed wesTMB captured the intended 90% of the in silico psTMB values. Although ∼1000× deep WES was applied, reference standard samples with less than 5% of tumor proportions are below the assay limit of detection (LoD) of wesTMB quantification. However, predicted wesTMB based on observed psTMB accurately classify (>0.97 AUC) for TMB high and low patient stratification even in samples with 2% of tumor content, which is more clinically relevant, as TMB determination should be a qualitative assay for TMB high and low patient classification. One targeted panel sequencing vendor using an optimized blood psTMB pipeline can further classify TMB status accurately (>0.82 AUC) in samples with only 1% of tumor content. Conclusions We developed a linear model to establish the quantitative correlation between wesTMB and psTMB. A set of DNA reference standards was produced in aid to standardize TMB measurements in samples with low tumor content across different targeted sequencing panels. This study is a significant contribution aiming to harmonize TMB estimation and extend its future application in clinical samples with low tumor content including liquid biopsy.

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Introduction: Recent studies exhibited the unstable prediction ability of blood-based tumor mutational burden (bTMB) when predicting the response of immune checkpoint inhibitors (ICIs) therapy in patients with non-small cell lung cancer (NSCLC). Circulating tumor DNA (ctDNA) abundance, usually represented by maximum somatic allele frequency (MSAF), was one possible confounding factor influencing bTMB ability in ICIs response prediction. Methods: MSAF-adjusted bTMB (Ma-bTMB) was established and validated in patients with advanced NSCLC among Geneplus Cancer Genome Database (GCGD, n = 1679), Zhuo (n = 35), Wang (n = 45), POPLAR (NCT01903993, n = 211) and OAK (NCT02008227, n = 642) cohorts. Results: MSAF demonstrated a modest positive correlation with bTMB and a negative one with survival benefit. Improved survival outcomes of ICIs therapy have been observed among patients with high-Ma-bTMB compared to those with low-Ma-bTMB in Zhuo and Wang cohorts. In addition, compared to low-Ma-bTMB, high-Ma-bTMB was associated with more positive clinical benefits from ICIs therapy than chemotherapy both in POPLAR and OAK cohorts. Further exploration suggested that Ma-bTMB could precisely identify more potential ICIs beneficiaries compared to bTMB and LAF-bTMB, complementary to PD-L1 expression. Conclusions: We developed Ma-bTMB, a convenient, readily available, non-invasive predictive biomarker effectively differentiates beneficiaries of ICIs therapy in advanced NSCLC, warranting future clinical trials.