Low-frequency Mutations Research Articles

Comprehensive pan‑cancer analysis of potassium voltage-gated channel Q4 (KCNQ4) gene across multiple human malignant tumors

A large number of studies indicate that Potassium Voltage-Gated Channel Q4 (KCNQ4) gene is the cause of non-syndromic hearing loss, but there are few studies investigating the role of KCNQ4 in cancers and scarcity of comprehensive analysis of its involvement in the diagnosis, methylation, mutation, prognosis of various cancer types. Therefore, the aim of this study is to examine the anticancerous and immune effects of KCNQ4 in various cancers and its potential value in breast cancer. In this study, we explored the potential role of KCNQ4 in cancers using public databases and the R software for bioinformatics analysis. The results showed that the low expression of KCNQ4 across specific cancer types was positively associated with low mutation frequency and methylation, and the improved survival. Eight small molecule compounds were identified that could potentially target KCNQ4. In addition, immunohistochemistry confirmed that the KCNQ4 expression was low in breast cancer. In vitro experiments confirmed that overexpression of KCNQ4 inhibited cell migration and invasion and promoted apoptosis. In summary, our comprehensive pan-cancer analysis highlights the potential of KCNQ4 as a cancer marker, and can be used as an auxiliary prognostic indicator and an indicator for immunotherapy in certain tumor types.

Implications of combined NOD2 and other gene mutations in autoinflammatory diseases.

NOD-like receptors (NLRs) are intracellular sensors associated with systemic autoinflammatory diseases (SAIDs). We investigated the largest monocentric cohort of patients with adult-onset SAIDs for coinheritance of low frequency and rare mutations in NOD2 and other autoinflammatory genes. Sixty-three patients underwent molecular testing for SAID gene panels after extensive clinical workups. Whole exome sequencing data from the large Atherosclerosis Risk in Communities (ARIC) study of individuals of European-American ancestry were used as control. Of 63 patients, 44 (69.8%) were found to carry combined gene variants in NOD2 and another gene (Group 1), and 19 (30.2%) were carriers only for NOD2 variants (Group 2). The genetic variant combinations in SAID patients were digenic in 66% (NOD2/MEFV, NOD2/NLRP12, NOD2/NLRP3, and NOD2/TNFRSF1A) and oligogenic in 34% of cases. These variant combinations were either absent or significantly less frequent in the control population. By phenotype-genotype correlation, approximately 40% of patients met diagnostic criteria for a specific SAID, and 60% had mixed diagnoses. There were no statistically significant differences in clinical manifestations between the two patient groups except for chest pain. Due to overlapping phenotypes and mixed genotypes, we have suggested a new term, "Mixed NLR-associated Autoinflammatory Disease ", to describe this disease scenario. Gene variant combinations are significant in patients with SAIDs primarily presenting with mixed clinical phenotypes. Our data support the proposition that immunological disease expression is modified by genetic background and environmental exposure. We provide a preliminary framework in diagnosis, management, and interpretation of the clinical scenario.

Competitive interactions facilitate resistance development against antimicrobials.

While the evolution of antimicrobial resistance is well studied in free-living bacteria, information on resistance development in dense and diverse biofilm communities is largely lacking. Therefore, we explored how the social interactions in a duo-species biofilm composed of the brewery isolates Pseudomonas rhodesiae and Raoultella terrigena influence the adaptation to the broad-spectrum antimicrobial sulfathiazole. Previously, we showed that the competition between these brewery isolates enhances the antimicrobial tolerance of P. rhodesiae. Here, we found that this enhanced tolerance in duo-species biofilms is associated with a strongly increased antimicrobial resistance development in P. rhodesiae. Whereas P. rhodesiae was not able to evolve resistance against sulfathiazole in monospecies conditions, it rapidly evolved resistance in the majority of the duo-species communities. Although the initial presence of R. terrigena was thus required for P. rhodesiae to acquire resistance, the resistance mechanisms did not depend on the presence of R. terrigena. Whole genome sequencing of resistant P. rhodesiae clones showed no clear mutational hot spots. This indicates that the acquired resistance phenotype depends on complex interactions between low-frequency mutations in the genetic background of the strains. We hypothesize that the increased tolerance in duo-species conditions promotes resistance by enhancing the selection of partially resistant mutants and opening up novel evolutionary trajectories that enable such genetic interactions. This hypothesis is reinforced by experimentally excluding potential effects of increased initial population size, enhanced mutation rate, and horizontal gene transfer. Altogether, our observations suggest that the community mode of life and the social interactions therein strongly affect the accessible evolutionary pathways toward antimicrobial resistance.IMPORTANCEAntimicrobial resistance is one of the most studied bacterial properties due to its enormous clinical and industrial relevance; however, most research focuses on resistance development of a single species in isolation. In the present study, we showed that resistance evolution of brewery isolates can differ greatly between single- and mixed-species conditions. Specifically, we observed that the development of antimicrobial resistance in certain species can be significantly enhanced in co-culture as compared to the single-species conditions. Overall, the current study emphasizes the need of considering the within bacterial interactions in microbial communities when evaluating antimicrobial treatments and resistance evolution.

Susceptibility of pediatric acute lymphoblastic leukemia to STAT3 inhibition depends on p53 induction.

Advances in the clinical management of pediatric B-cell acute lymphoblastic leukemia (B-ALL) have dramatically improved outcomes for this disease. However, relapsed and high-risk disease still contribute to significant numbers of treatment failures. Development of new, broad range therapies is urgently needed for these cases. We previously reported the susceptibility of ETV6-RUNX1+ pediatric B-ALL to inhibition of signal transducer and activator of transcription 3 (STAT3) activity. In the present study, we demonstrate that pharmacological or genetic inhibition of STAT3 results in p53 induction and that CRISPR-mediated TP53 knockout substantially reverses susceptibility to STAT3 inhibition. Furthermore, we demonstrate that sensitivity to STAT3 inhibition in patient-derived xenograft (PDX) B-ALL samples is not restricted to any particular disease subtype, but rather depends on TP53 status, the only resistant samples being TP53 mutant. Induction of p53 following STAT3 inhibition is not directly dependent on MDM2 but correlates with degradation of MDM4. As such, STAT3 inhibition exhibits synergistic in vitro and in vivo anti-leukemia activity when combined with MDM2 inhibition. Taken together with the relatively low frequency of TP53 mutations in this disease, these data support the future development of combined STAT3/ MDM2 inhibition in the therapy of refractory and relapsed pediatric B-ALL.

Integrated Multi-Omics Profiling of Young Breast Cancer Patients Reveals a Correlation between Galactose Metabolism Pathway and Poor Disease-Free Survival.

In recent years, there has been a notable rise in the incidence of breast cancer among young patients, who exhibit worse survival outcomes and distinct characteristics compared to intermediate and elderly patients. Therefore, it is imperative to identify the specific features unique to young patients, which could offer insights into potential therapeutic strategies and improving survival outcomes. In our study, we performed an integrative analysis of bulk transcriptional and genomic data from extensive clinical cohorts to identify the prognostic factotrs. Additionally, we analyzed the single-cell transcriptional data and conducted in vitro experiments. Our work confirmed that young patients exhibited higher grading, worse disease-free survival (DFS), a higher frequency of mutations in TP53 and BRCA1, a lower frequency of mutations in PIK3CA, and upregulation of eight metabolic pathways. Notably, the galactose metabolism pathway showed upregulation in young patients and was associated with worse DFS. Further analysis and experiments indicated that the galactose metabolism pathway may regulate the stemness of cancer cells and ultimately contribute to worse survival outcomes. In summary, our finding identified distinct clinicopathological, transcriptional, and genomics features and revealed a correlation between the galactose metabolism pathway, stemness, and poor disease-free survival of breast cancer in young patients.

Fast, sensitive, and specific multiplexed single-molecule detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) analysis has emerged as a highly promising non-invasive assay for detection and monitoring of cancer. However, identification of multiple point-mutant ctDNAs, particularly at extremely low frequencies in early cancer stages, remains a significant challenge. To address this issue, we present a multiplexed ctDNA detection technique, SIMUL (single-molecule detection of multiple low-frequency mutations). SIMUL involves an unbiased preamplification of both wild-type and mutant DNAs, followed by the detection of mutant DNAs through single-molecule multicolor imaging. SIMUL enables highly sensitive and specific detection of multiple single-nucleotide mutations in a short span of time, even in the presence of 10,000-fold excess of wild-type DNA. Importantly, SIMUL can accurately measure mutant fractions due to its linear correlation between the number of single-molecule spots and the variant allele frequency. This breakthrough technique holds immense potential for clinical applications, offering significant improvements for example in early cancer detection and accurate evaluation of anticancer treatment responses.

First Report of a Low-Frequency Mosaic Mutation in the Hydroxymethylbilane Synthase Gene Causing Acute Intermittent Porphyria.

Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient's blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics.

Exosomal NOX1 promotes tumor-associated macrophage M2 polarization-mediated cancer progression by stimulating ROS production in cervical cancer: a preliminary study

BackgroundCervical cancer the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women, with an estimated 604,000 new cases and 342,000 deaths worldwide in 2020 for high rates of recurrence and metastasis. Identification of novel targets could aid in the prediction and treatment of cervical cancer. NADPH oxidase 1 (NOX1) gene-mediated production of reactive oxygen species (ROS) could induce migration and invasion of cervical cancer cells. Tumor-associated macrophages (TAMs) play important roles in cervical cancer. Tumor cell-derived exosomes mediate signal transduction between the tumor and tumor microenvironment. Elucidation of the mechanisms of NOX1-carrying exosomes involved in the regulation of TAMs may provide valuable insights into the progression of cervical cancer.MethodsUniformly standardized mRNA data of pan-carcinoma from the UCSC database were downloaded. Expression of NOX1 in tumor and adjacent normal tissues for each tumor type was calculated using R language software and significant differences were analyzed. SNP data set were downloaded for all TCGA samples processed using MuTect2 software from GDC. Cell experiment and animal tumor formation experiment were used to evaluate whether exosomal NOX1 stimulating ROS production to promote M2 polarization of TAM in cervical cancer.ResultsNOX1 is highly expressed with a low mutational frequency in pan-carcinoma. Upregulation of NOX1 may be associated with infiltration of M2-type macrophages in cervical cancer tissues, and NOX1 promotes malignant features of cervical cancer cells by stimulating ROS production. Exosomal NOX1 promotes M2 polarization of by stimulating ROS production. Exosomal NOX1 enhances progression of cervical cancer and M2 polarization in vivo by stimulating ROS production.ConclusionExosomal NOX1 promotes TAM M2 polarization-mediated cancer progression through stimulating ROS production in cervical cancer.

Use of whole genome sequencing to identify low-frequency mutations in SARS-CoV-2 patients treated with remdesivir.

Remdesivir (RDV) has been shown to reduce hospitalization and mortality in COVID-19 patients. Resistance mutations caused by RDV are rare and have been predominantly reported in patients who are on prolonged therapy and immunocompromised. We investigate the effects of RDV treatment on intra-host SARS-CoV-2 diversity and low-frequency mutations in moderately ill hospitalized COVID-19 patients and compare them to patients without RDV treatment. From March 2020 to April 2022, sequential collections of nasopharyngeal and mid-turbinate swabs were obtained from 14 patients with and 30 patients without RDV treatment. Demographic and clinical data on all patients were reviewed. A total of 109 samples were sequenced and mutation analyses were performed. Previously reported drug resistant mutations in nsp12 were not identified during short courses of RDV therapy. In genes encoding and surrounding the replication complex (nsp6-nsp14), low-frequency minority variants were detected in 7/14 (50%) and 18/30 (60%) patients with and without RDV treatment, respectively. We did not detect significant differences in within-host diversity and positive selection between the RDV-treated and untreated groups. Minimal intra-host variability and stochastic low-frequency variants detected in moderately ill patients suggests little selective pressure in patients receiving short courses of RDV. The barrier to RDV resistance is high in patients with moderate disease. Patients undergoing short regimens of RDV therapy should continue to be monitored.

Dengue virus serotypic replacement of NS3 protease or helicase domain causes chimeric viral attenuation but can be recovered by a compensated mutation at helicase domain or NS2B, respectively.

Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%-78% amino acid identities, resulting in incomplete immunity, where one serotype's infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5' RNA triphosphatase (5'RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations' frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells. IMPORTANCE Enzyme activities for replicating DENV 5' cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5' cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)-NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.

DELFMUT: duplex sequencing-oriented depth estimation model for stable detection of low-frequency mutations.

Duplex sequencing technology has been widely used in the detection of low-frequency mutations in circulating tumor deoxyribonucleic acid (DNA), but how to determine the sequencing depth and other experimental parameters to ensure the stable detection of low-frequency mutations is still an urgent problem to be solved. The mutation detection rules of duplex sequencing constrain not only the number of mutated templates but also the number of mutation-supportive reads corresponding to each forward and reverse strand of the mutated templates. To tackle this problem, we proposed a Depth Estimation model for stable detection of Low-Frequency MUTations in duplex sequencing (DELFMUT), which models the identity correspondence and quantitative relationships between templates and reads using the zero-truncated negative binomial distribution without considering the sequences composed of bases. The results of DELFMUT were verified by real duplex sequencing data. In the case of known mutation frequency and mutation detection rule, DELFMUT can recommend the combinations of DNA input and sequencing depth to guarantee the stable detection of mutations, and it has a great application value in guiding the experimental parameter setting of duplex sequencing technology.

HIV-1 Low-Frequency Variants Identified in Antiretroviral-Naïve Subjects with Virologic Failure after 12 Months of Follow-Up in Panama.

Low-frequency mutations associated with drug resistance have been related to virologic failure in subjects with no history of pre-treatment and recent HIV diagnosis. In total, 78 antiretroviral treatment (ART)-naïve subjects with a recent HIV diagnosis were selected and followed by CD4+ T lymphocytes and viral load tests to detect virologic failure. We sequenced the basal samples retrospectively using next-generation sequencing (NGS), looking for low-frequency mutations that had not been detected before using the Sanger sequencing method (SSM) and describing the response to ART. Twenty-two subjects developed virologic failure (VF), and thirteen of them had at least one drug-resistance mutation associated with Reverse Transcriptase Inhibitors (RTI) and Protease Inhibitors (PIs) at frequency levels ≤ 1%, not detected previously in their basal genotyping test. No resistance mutations were observed to Integrase Strand Transfer Inhibitors (INSTIs). We identified a possible cause of VF in ART-naïve subjects with low-frequency mutations detected. To our knowledge, this is the first evaluation of pre-existing drug resistance for HIV-1 minority variants carried out on ART-naïve people living with HIV/AIDS (PLWHA) by analyzing the HIV-1 pol gene using NGS in the country.

CRISPincette, and detection of low frequency cancer mutations in ctDNA.

61 Background: Liquid biopsy has emerged as a non-invasive and widely applicable approach in clinical practice for cancer detection and monitoring. Detecting low-frequency mutations in circulating tumor DNA (ctDNA) is crucial for the success of liquid biopsy; however, technical limitations have hindered the detection of low variant allele frequency (VAF) mutations. To address this issue, we developed CRISPincette, a novel method which combines next-generation sequencing with an advanced CRISPR system, GeneCker-Cas9. Methods: CRISPincette was designed to efficiently detect mutant alleles by eliminating abundant wild-type alleles from normal cell-induced cell-free DNA (cfDNA) using CRISPR/Cas9. An engineered CRISPR/Cas9 variant, GeneCker-Cas9, was developed to maintain single-base precision outside the PAM region, enabling efficient discrimination of single-base mutations at all 20 positions within a single guide RNA target sequence. This advanced fidelity significantly improved the accuracy compared to high-specificity SpCas9 variants (SpCas9-HF and eSpCas9). To validate the sensitivity and accuracy of ctDNA detection using CRISPincette, we employed NGS standard materials (Horizon HD776, 778, 779) and assessed the improvement in ctDNA detection sensitivity for EGFR gene mutations (EGFR 19del, EGFR L858R, EGFR T790M). Subsequently, we analyzed the clinical applicability of CRISPincette using plasma samples from 7 non-small cell lung cancer (NSCLC) patients (6 patients with EGFR mutations and 1 patient without EGFR mutations). Results: Our analysis showed an approximately 10 to 30-fold enhancement in the detection sensitivity of EGFR gene mutations. Concordant detection of EGFR mutations was observed in the 6 patients with EGFR mutations using real-time PCR tests in each tissue biopsy, and no EGFR mutation was detected in the patient without the EGFR mutation. This confirmed CRISPincette's high accuracy and sensitivity in detecting low-frequency mutations in ctDNA. Conclusions: The implementation of CRISPincette has significantly improved the accuracy and sensitivity of liquid biopsy, making it a reliable and non-invasive alternative for confirming molecular pathological information in cancer patients. Furthermore, the high accuracy of CRISPincette will be a powerful tool for monitoring treatment and prognosis through the timing of anticancer drug administration. Overall, CRISPincette represents a substantial advancement in liquid biopsy technology, with potential implications for cancer diagnosis, treatment allocation, and monitoring the emergence of resistance.

Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

Lethal and mutagenic effects of different LET radiations on Bacillus subtilis spores

New, useful microorganism resources have been generated by ionizing radiation breeding technology. However, the mutagenic effects of ionizing radiation on microorganisms have not been systematically clarified. For a deeper understanding and characterization of ionizing radiation-induced mutations in microorganisms, we investigated the lethal effects of seven different linear energy transfer (LET) radiations based on the survival fraction (SF) and whole-genome sequencing analysis of the mutagenic effects of a dose resulting in an SF of around 1% in Bacillus subtilis spores. Consequently, the lower LET radiations (gamma [surface LET: 0.2 keV/µm] and 4He2+ [24 keV/µm]) showed low lethality and high mutation frequency (MF), resulting in the major induction of single-base substitutions. Whereas higher LET radiations (12C5+ [156 keV/µm] and 12C6+ [179 keV/µm]) showed high lethality and low MF, resulting in the preferential induction of deletion mutations. In addition, 12C6+ (111) ion beams likely possess characteristics of both low- and high-LET radiations simultaneously. A decrease in the relative biological effectiveness and an evaluation of the inactivation cross section indicated that 20Ne8+ (468 keV/µm) and 40Ar13+ (2214 keV/µm) ion beams had overkill effects. In conclusion, in the mutation breeding of microorganisms, it should be possible to regulate the proportions, types, and frequencies of induced mutations by selecting an ionizing radiation of an appropriate LET in accordance with the intended purpose.

Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency.

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200µM) and DNA was sequenced 48, 72, and 96h post-exposure. A DS mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A significant increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (r=0.97). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The consistent results across the three time points suggest that 48h may be sufficient for mutation analysis post-exposure. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DS for characterizing chemical mutagenicity in both research and regulatory evaluation.

Characterization of Two Novel Inhibitors of the Mycobacterium tuberculosis Cytochrome bc1 Complex.

Drug-resistant tuberculosis is a global health care threat calling for novel effective treatment options. Here, we report on two novel cytochrome bc1 inhibitors (MJ-22 and B6) targeting the Mycobacterium tuberculosis respiratory chain with excellent intracellular activities in human macrophages. Both hit compounds revealed very low mutation frequencies and distinct cross-resistance patterns with other advanced cytochrome bc1 inhibitors.

Evaluation of the Prognostic Value of Low-Frequency KRAS Mutation Detection in Circulating Tumor DNA of Patients with Metastatic Colorectal Cancer.

KRAS mutation in tumor tissue is a well-known predictor of resistance to the treatment of anti-EGFR antibodies in metastatic colorectal cancers (mCRC). However, the prognostic value of low-frequency plasma circulating tumor DNA (ctDNA) KRAS mutation in predicting treatment resistance in pretreated mCRC patients remains controversial. This study retrospectively reviewed the clinical course, including response to anti-EGFR and anti-VEGF therapies, and changes in serum tumor marker levels along with image studies in mCRC patients with <1.5% KRAS mutations detected in plasma ctDNA by next-generation sequencing (NGS) at a single center in Taiwan. We identified six pretreated mCRC patients with low-frequency KRAS G12V/G12D/G12S/G13D mutations (variant allele frequency 0.26~1.23%) in plasma ctDNA. Co-occurring low-frequency ctDNA mutations in APC, TP53, MAP2K1, KEAP1, or CTNNB1 were also detected. Although all six patients had treatment adjustments within one month after the ctDNA genetic test, image-evident tumor progression was noted in all patients within a median of 4 months afterwards. Re-challenge therapy with a combination of anti-EGFR, anti-VEGF, and FOLFIRI chemotherapy was found to be ineffective in a patient with 0.38% KRAS G12D mutation in baseline ctDNA. Our study suggests that the detection of low-frequency KRAS mutations in ctDNA could be used as a predictor of treatment response in mCRC patients.

Subgrouping of Burkitt lymphoma variants by DNA methylation is driven by an EBV‐associated epigenotype

Introduction: Burkitt lymphoma (BL) is an aggressive mature B-cell lymphoma, which traditionally has been separated into three epidemiologic variants: endemic (eBL), sporadic (sBL) and immunodeficiency-associated BL (iBL). This subgrouping is strongly confounded by EBV-infection. Indeed, there is evidence from molecular studies that subtyping of BL based on EBV status might better reflect the pathogenetic heterogeneity than epidemiology. As EBV is known to influence epigenetic regulation, we here aimed at a comparative DNA methylation (DNAme) profiling of BL variants with respect to their geographic origin and EBV status. Methods: We collected DNAme profiles (Infinium HumanMethylation450 and Infinium MethylationEPIC BeadChip) of 116 BL patients (80 sBL: 7 EBV+; 29 eBL: 27 EBV+; 7 iBL: 4 EBV+), 17 BL cell lines and 6 EBV-transformed LCLs. For group determination unsupervised clustering algorithms (K-means, PGMRA, UMAP) were applied. To investigate potential consequences of EBV-associated DNAme we performed a protein-protein interaction network analysis on genes within the EBV-specific DNAme pattern. Moreover, we mined gene expression values from RNAseq in 21 EBV- sBL and 5 germinal-center B-cell populations. Results: Unsupervised clustering algorithms revealed two distinct DNAme phenotypes, which differentiate EBV+ and EBV- BL based on 1,266 CpGs. This separation is accompanied by a characteristic hypermethylation in EBV+ BLs. Additionally, we identified a subgroup of EBV+ BL with a similar DNAme pattern like the LCLs, possibly indicating latency phase differences within EBV+ BLs. Investigation of the EBV-specific DNAme pattern revealed a network with UBC as a key interactor, indicating the potential importance of the ubiquitin-proteasome system (UPS) in EBV-induced lymphomagenesis. Among genes differentially methylated between EBV+ and EBV- BL, 19 genes have been reported as recurrently mutated in BL. All except 3 of these genes are expressed in sBL and germinal-center B-cells. All these genes were significantly hypermethylated within regulatory regions in EBV+ BLs (σ/σmax = 0.4, q ≤ 0.01), including genes with a lower mutational frequency in EBV+ versus EBV- BLs. Conclusion: The findings of this study show the EBV status to be strongly associated with DNAme subgroups in BL, supporting the subtyping of BL variants based on their EBV status rather than their geographic origin. Moreover, the pattern of DNAme in EBV+ BL might suggest epigenetic silencing and deregulated ubiquitination as means alternative to gene mutation to be involved in the pathogenesis of EBV+ BL. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Genomics, Epigenomics, and Other -Omics, Pathology and Classification of Lymphomas No conflicts of interests pertinent to the abstract.

Comparison of mutation detection rate between cell pellet DNA and cell-free DNA from cerebrospinal fluid in patients with advanced lung cancer.

e21014 Background: Liquid biopsy of cerebrospinal fluid (CSF) has been approved to be a promising strategy for molecular pathology testing in patients with lung cancer. Numerous studies showed that CSF-derived DNA can recapitulate the molecular characteristics of metastatic lung cancer. However, differences in mutation detection rate and the overall mutational profile between cell pellet DNA and supernatant cell-free DNA (cfDNA) from CSF in patients with lung cancer have rarely been reported. Methods: Matched CSF cell pellet and supernatant sample sets were selected from 51 lung cancer patients in our sequencing center. CSF was centrifuged (1000g, 10 min) to separate the cell pellet and supernatant components. Cellular DNA and cfDNA were extracted and underwent library preparation procedures for next-generation sequencing respectively. Cut adapter, mapping, fusion calling and cnv calling were performed for sequencing data analysis. Results: At the sample level, gene mutation could be detected in both CSF cell pellet and cfDNA, and the positive detection rate of gene mutation was consistent. The positive percent agreement (PPA) of CSF cfDNA sequencing compared with CSF cell pellet DNA was 93%. At the genetic variation level, 223 gene mutation sites were detected, and the positive rate of gene mutation in the CSF cfDNA (32.51%) was significantly higher than that in the CSF cell pellet (10.74%, p＜0.01). Additionally，the variation rate of less than 1% was defined as the low-frequency mutation range. Results showed that variant allele frequency (VAF) in CSF cfDNA were remarkably higher than those in the CSF cell pellet in both low-and high-frequency mutation ranges ( p＜0.01, p＜0.01). Conclusions: Both cell pellet DNA and cfDNA isolated from CSF can be used for molecular subtyping of lung cancer. Given that CSF is inevitably contaminated with blood cells in clinical procedures, the dynamic range of tumor cells in CSF could be quite large. CSF cfDNA better recapitulate the mutational status and is recommended to consider first for liquid biopsy in advanced lung cancer with brain metastases or suspicious brain metastases. Key words: Next-generation sequencing; CSF; cell pellet DNA; cfDNA; advanced lung cancer.