Dynamic Expedition of Leading Mutations in SARS-CoV-2 Spike Glycoproteins

During the ongoing CoVID-19 epidemic, the continuous genomic evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been generating new variants with enhanced transmissibility and immune escape. Being one key target of antibodies, mutations of the spike glycoprotein play a vital role in the trajectory of virus evasion. Here, we present a time-resolved statistical method, dynamic expedition of leading mutations (deLemus), to analyze the evolution dynamics of the spike protein. Together with analysis on single amino-acid polymorphism (SAP), we proposed one L-index to quantify the mutation strength of each amino acid for unravelling mutation pattern of spike glycoprotein. The sites of interest (SOI) with high L-index hold great promise to detect potential signal of emergent variants.

MPC-5 Characteristics of H3 G34-mutant gliomas

Introduction: Diffuse hemispheric gliomas, H3 G34-mutant (DHG H3G34-mutant) are newly recognized infiltrating gliomas of the cerebral hemispheres of pediatric and young adult patients. We experienced 6 DHG H3G34-mutant cases. In this study, we describe the clinical, radiological and pathological characteristics of these cases. Result: Mean age at diagnosis was 16.8 years (range:10–26). Three patients were male. Among six cases, tumors located in cerebral cortex in five cases and multiple sites including basal ganglia and cortex in a case. All tumors showed no or only a faint contrast-enhancement and harbored restriction of diffusion. One patient underwent total resection, four underwent partial resection and one underwent biopsy. Pathological diagnosis were CNS embryonal tumors (n=3/6), glioblastoma, IDH-wildtype (n=2/6) and anaplastic astrocytoma, IDH-wildtype (n=1/5). All cases were negative for Olig2 and positive for GFAP in immunohistochemistry. Mean Ki-67 index was 38% (range: 10–60%). All cases revealed at least one of mitosis, necrosis or microvascular proliferation. Especially, mitosis was the most frequently found (n=5/6). The H3F3A mutations were G34R mutations in all cases. One case revealed a characteristic mutation pattern, therefore now we are performing further examination. Adjuvant chemoradiotherapies were performed for all cases. Mean progression free survival was 10.1 months (range: 1.6–33.1). Discussion: As published literatures reported, all cases exhibited restriction of diffusion and negative for Olig2. For a cerebral hemispheric tumor of pediatric or young adult patient which shows restriction of diffusion and no contrast-enhancement, and of which pathological findings is malignant and olig2 is negative, genetic analysis of H3F3A gene might be essential.

Hotspots for mutations in the SARS-CoV-2 spike glycoprotein: a correspondence analysis

Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the SgpSARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.

Differential impact of IDH1/2 mutational subclasses on outcome in adult AML: Results from a large multicenter study

Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are amongst the most frequent alterations in acute myeloid leukemia (AML) and can be found in ~20% of patients at diagnosis. Among 4930 patients (median age 56 years, interquartile range 45-66) with newly diagnosed, intensively treated AML, we have identified IDH1 mutations (mIDH1) in 423 (8.6%) and IDH2 mutations (mIDH2) in 575 (11.7%) patients. Overall, there were no differences in response rates or survival for patients with mIDH1 or mIDH2 compared to patients without mutated IDH1/2. However, distinct clinical and co-mutational phenotypes of the most common subtypes of IDH1/2 mutations could be associated with differences in outcome. IDH1-R132C was associated with significantly increased age, lower white blood cell count (WBC), less frequent co-mutation of NPM1 and FLT3-ITD as well as lower rate of complete remissions and a trend for reduced overall survival (OS) compared to other mIDH1 variants and wtIDH1/2. In our analysis, IDH2-R172K was associated with significantly lower WBC, more karyotype abnormalities, and less frequent co-mutations of NPM1 and/or FLT3-ITD. Among patients within the ELN2017 intermediate- and adverse-risk groups, RFS and OS were significantly better for patients with IDH2-R172K compared to wtIDH, providing evidence that AML with IDH2-R172K could be a distinct entity with a specific co-mutation pattern and favorable outcome. In summary, the presented data from a large cohort of IDH1/2 mutant AML patients indicate novel and clinically relevant findings for the most common IDH-mutation subtypes.

Comprehensive Analysis of TP53 Mutation Characteristics and Identification of Patients with Inferior Prognosis and Enhanced Immune Escape in Diffuse Large B Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphatic malignancy from both the clinical and molecular standpoints. TP53 mutations have been observed in DLBCL with high frequency mutations, which play a crucial role in lymphomagenesis. Considering the complex pathophysiological mechanisms involved in DLBCL, we herein assume that the interaction of TP53 with other genetic variants together promote the development of DLBCL. We also explore that these genetic interactions shape the discrepant immune landscape in DLBCL. Methods: We performed a comprehensive analysis of TP53 mutations in the DNA-binding domain (DBD) region. Genome alterations that were mutually exclusive or co-occurring with TP53 mutations were identified, and their potential value in prognosis and possible relationships with tumor microenvironment (TME) were further examined using targeted next-generation sequencing (n=176), transcriptome sequencing (n=152) and circulating tumor DNA sequencing (n=38). Results: TP53 was frequently mutated in DLBCL with a rate of 30% (53 of 176). The occurrence of TP53 mutations was skewed toward the early stages of DLBCL pathogenesis. Among these variants of TP53, 74% were missense mutations, and the remaining were inactivating frameshift indels, nonsense mutations, coding sequencing indels and splicing mutations. Importantly, most mutations (87.5%) occurred in exons 5-8, which encoded the DNA-binding domain (DBD) region of TP53. Codons 175, 273, and 248 of the p53 protein had the highest mutation frequency, which are also the hot spots of TP53 mutation found in most human cancers. However, TP53 alone is insufficient to effectively differentiate the risk of DLBCL, even when only considering mutations in the DBD region. CD58 mutations, which are mutually exclusive from TP53 mutations, in combination with TP53 mutations, could significantly differentiate the prognosis of DLBCL. The survival of patients with either one of the mutually exclusive mutation patterns, namely, TP53MUT&CD58WT or TP53WT&CD58MUT, was inferior to those harboring both wild-type TP53 and CD58. Notably, patients with TP53WT&CD58MUT mutation pattern had the worst outcome and were characterized by a higher overall tumor mutation burden (TMB) and immune score. An enhanced immune escape feature, including the abundant infiltration of inflammatory cells and upregulation of inhibitory immunomodulatory molecules such as PD-1, TIM3, LAG3, KLRC1, KLRC3, KLRD1, IDO1, IDO2 and TDO2, was observed in these patients with TP53WT&CD58MUT mutation pattern. A larger number of terms related to cytokine and chemokine and inflammatory pathways were enriched in the TP53WT&CD58MUT group, including cytokine and chemokine production, binding and activity, and interferon-γ pathway. Additionally, GSEA demonstrated that interferon-α and -γ responses and IL-6/JAK/STAT3 signaling enriched in the TP53WT&CD58MUT group. These patients with TP53WT&CD58MUT mutation pattern represent the candidate populations for immune therapy. Conclusions: Our findings indicated that the mutation patterns of TP53 and CD58 accurately stratified patients with DLBCL to permit the optional immunotherapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Endometrial Adenocarcinoma Biopsies: Can Targeted Education Increase Reproducibility and Is It a Good Idea?

Introduction/Objective Assessing p53 mutation on biopsies could provide prognostic and therapeutic planning information, as it predicts worse outcomes for patients with endometrioid adenocarcinoma (EA). Immunohistochemistry (IHC) mutation pattern staining varies, easily causing misinterpretation. This study was designed to see if education clarifying staining issues would increase reproducibility, and if mutations detected in this setting were of prognostic import. Methods/Case Report p53 IHC on 46 FIGO grade 1 EA biopsies was scored by blinded participants. Education was provided regarding internal controls (IC), and common misinterpretations. The participants rescored to assess reproducibility. Outcome parameters (clinical stage [CS], progression free survival, higher FIGO grade on resection, and presence of mismatch repair mutations) were also assessed. Results (if a Case Study enter NA) 25% of scores changed post-education. Intraclass correlation among raters was 0.29 pre and 0.43 post (from fair to moderate). Mutation status changed in 5 cases. 2/5 caught IC failure for 1 case. Post-participation surveys found 0 participants were previously familiar with “high wild type,” and 60% were unfamiliar with cytoplasmic staining pattern, the possibility for heterogeneity, and necessary ICs. Every participant agreed grading was easier following education. Out of 6 patients with high CS disease (IIIC-IV), none had p53 mutation. 2/11 cases with higher FIGO grade (2-3) on resection had mutation. One patient had recurrence with no detectable mutation. 15/46 patients had MMR results available. No p53 mutation was detected in those with a loss of MLH1 and PMS2 (n=8). Conclusion Education on IHC issues can increase reproducibility in scoring, though overall reproducibility on biopsies was still subpar. Mutation detected in this setting did not correlate with current outcome parameters. Given the possibility of heterogenous expression and difficulties interpreting edge effect and assessing ICs in scant specimens, p53 IHC on EA biopsies is not recommended.

Molecular Evolution and Epidemiological Characteristics of SARS COV-2 in (Northwestern) Poland

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolved into a worldwide outbreak, with the first Polish cases in February/March 2020. This study aimed to investigate the molecular epidemiology of the circulating virus lineages between March 2020 and February 2021. We performed variant identification, spike mutation pattern analysis, and phylogenetic and evolutionary analyses for 1106 high-coverage whole-genome sequences, implementing maximum likelihood, multiple continuous-time Markov chain, and Bayesian birth–death skyline models. For time trends, logistic regression was used. In the dataset, virus B.1.221 lineage was predominant (15.37%), followed by B.1.258 (15.01%) and B.1.1.29 (11.48%) strains. Three clades were identified, being responsible for 74.41% of infections over the analyzed period. Expansion in variant diversity was observed since September 2020 with increasing frequency of the number in spike substitutions, mainly H69V70 deletion, P681H, N439K, and S98F. In population dynamics inferences, three periods with exponential increase in infection were observed, beginning in March, July, and September 2020, respectively, and were driven by different virus clades. Additionally, a notable increase in infections caused by the B.1.1.7 lineage since February 2021 was noted. Over time, the virus accumulated mutations related to optimized transmissibility; therefore, faster dissemination is reflected by the second wave of epidemics in Poland.

Ciprofloxacin Induced Antibiotic Resistance in Salmonella Typhimurium Mutants and Genome Analysis

Salmonella species is an important food-borne pathogen. Ciprofloxacin is used for treatment of salmonellosis. The wild type of Salmonella Typhimurium was exposed to 10-fold of minimal inhibitory concentration (MIC) of ciprofloxacin. The survivors of the first exposure were regrown. This regrown population was exposed to similar ciprofloxacin treatment. After 20 repetitions of the antibiotic exposure, the survival population was evaluated for antibiotic resistance. The mutants had 100-fold more of the initial MIC. The mutants were of smaller size (0.7 ± 0.5 mm) with a lag phase of 70 min. These mutants were resistant to other antibiotics belonging to different classes but were sensitive to heat and osmotic stress. The whole genome sequencing (WGS) analysis of antibiotic resistant mutants revealed interesting mutation pattern. Total 40513 mutations were observed across the genome (total 3843 annotated genes). Relatively small percentage (5.2%) of mutations were non-synonymous. Four-fold more transitions were observed than transversions. Ratio of <1 transition vs transversion shows a positive selection for antibiotic resistant trait. Seven genes with over 50 mutations were observed. However, mutation distribution across the genome was relatively uniform. The important genes like dnaE, gyrA, iroC, metH and rpoB involved in antibiotic resistance had point mutations. The genome analysis showed that most affected pathways were transcription regulation, transmembrane transport, cell adhesion, pathogenesis, pilus assembly, oxidation-reduction mechanisms, phosphor-relay signal transduction and LPS biosynthesis. We propose that development resistance to ciprofloxacin is because of the mutations in these genes.