Increase In Mutation Frequency Research Articles

In vitro and in vivo assessments of the genotoxic potential of 3-chloroallyl alcohol.

3-Chloroallyl alcohol (3-CAA) can be found in the environment following the application of plant protection products. 3-CAA is formed in groundwater following the injection of 1,3-dichloropropene, a fumigant used to control nematodes. 3-CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3-CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3-CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3-CAA was negative in an acute in vivo CD-1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3-CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3-CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3-CAA is not considered to pose a mutagenic risk.

Multiple Myeloma Evolution Is Characterized By Dynamic Epigenetic Landscapes

The biological mechanisms driving MM progression and multi-drug resistance remain unclear. To better characterize the molecular mechanisms driving malignant transformation and disease progression, we generated a new database comprising 1,016 bone marrow biopsies from patients across the whole MM spectrum. These samples were clinically, genetically (844 RNA-Seq, 870 WES, 7 scRNA-Seq), and epigenetically (11 scATAC-Seq, 8 CUT&Tag) characterized. While we have observed increased mutation frequency in MM driver genes in later disease stages, no specific mutations statistically correlated with malignant transformation, nor with the development of refractory disease. Enrichment analysis of transcriptional data identified biological pathways and underlying molecular events associated with MM progression and therapy resistance: cell adhesion, inflammatory cytokines, TME-dependence and hematopoietic cell identity were under-expressed in NDMM vs. premalignant disease; whereas cell cycle, energy metabolism, DNA repair, and protein/RNA synthesis/degradation were overexpressed in LRMM compared to NDMM. Importantly, this transcriptional dysregulation was anecdotally confirmed in patients with multiple sequential samples. To identify the main biological pathways responsible for the inter-sample transcriptomic variability of our cohort, we performed a principal component analysis (PCA) using single sample gene set enrichment analysis (ssGSEA) NES of Cancer Hallmarks. The first component (PC1) was associated with aforementioned hallmarks differentially expressed in NDMM and LRMM samples. This suggested PC1 as a possible prognostic biomarker, which was confirmed by Cox Proportional Hazard models, associated with shorter TTP in SMOL, PFS in NDMM, and OS in active MM. To elucidate the mechanisms driving such aberrant transcriptional programs, we applied a robust dimensionality reduction technique (t-SNE) and a clustering method (fuzzy c-means) to RNA-Seq data to identify clusters of co-expressing genes, thus characterizing MM transcriptomic topology. In total, 16,738 genes were grouped in 500 gene clusters putatively controlled by shared transcriptional programs. Enrichment analysis showed that gene clusters under-expressed during malignant transformation were enriched for H3K27me3, whereas gene clusters overexpressed in refractory disease were enriched for H3K27ac. We validated these findings using scATAC-Seq in 11 MM samples (1 MGUS, 3 SMOL, 4 NDMM, 1 ERMM and 2 LRMM), which confirmed that a greater decrease in chromatin accessibility occurred among the most under-expressed gene clusters during malignant transformation. scATAC-Seq data also revealed the differential accessibility of B cell master regulators' binding motifs across disease progression (e.g., SPI1 in MGUS, FOS in SMOL, the IRF and POU family in NDMM, RUNX2 and RFX5 in LRMM) and suggested MM pioneer TFs by correlating the accessibility of TF's genes to their targets' expression (e.g., SPI1, BACH2, RELA, IRF4, among others). CUT&Tag data from 4 NDMM and 4 LRMM samples confirmed that the strongest H3K27ac signal was found in the topological region overexpressed in refractory disease, with a positive correlation between H3K27ac signal and gene expression. Super enhancer (SE) analysis demonstrated that, while genes associated with active SEs showed higher level of expression than genes subjected to regular enhancers (RE) in all samples, LRMM presented increased expression of genes under both SE/RE control compared to their counterparts in NDMM. These observations, in conjunction with an increased expression of transcription regulation-associated DNA-binding proteins (DBPs; e.g., CDK9, CTCF, MED1, YY1), suggests that the aberrant gene expression in LRMM may be due to differential expression of these DBPs. In conclusion, MM progression, characterized by transcriptional dysregulation in response to TME and therapeutic stress, is epigenetically driven by DBPs whose activity is altered by genetic, cytogenetic, and epigenetic events. Future studies will determine the role of such masterregulators in MM biology and drug resistance, as well as allow us to propose patient-specific strategies for therapy re-sensitization.

Inhibition of ABL1 by tyrosine kinase inhibitors leads to a downregulation of MLH1 by Hsp70-mediated lysosomal protein degradation.

The DNA mismatch repair (MMR) pathway and its regulation are critical for genomic stability. Mismatch repair (MMR) follows replication and repairs misincorporated bases and small insertions or deletions that are not recognized and removed by the proofreading polymerase. Cells deficient in MMR exhibit an increased overall mutation rate and increased expansion and contraction of short repeat sequences in the genome termed microsatellite instability (MSI). MSI is often a clinical measure of genome stability in tumors and is used to determine the course of treatment. MMR is also critical for inducing apoptosis after alkylation damage from environmental agents or DNA-damaging chemotherapy. MLH1 is essential for MMR, and loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. In this study, we report that tyrosine kinase inhibitors, imatinib and nilotinib, lead to decreased MLH1 protein expression but not decreased MLH1 mRNA levels. Of the seven cellular targets of Imatinib and nilotinib, we show that silencing of ABL1 also reduces MLH1 protein expression. Treatment with tyrosine kinase inhibitors or silencing of ABL1 results in decreased apoptosis after treatment with alkylating agents, suggesting the level of MLH1 reduction is sufficient to disrupt MMR function. We also report MLH1 is tyrosine phosphorylated by ABL1. We demonstrate that MLH1 downregulation by ABL1 knockdown or inhibition requires chaperone protein Hsp70 and that MLH1 degradation can be abolished with the lysosomal inhibitor bafilomycin. Taken together, we propose that ABL1 prevents MLH1 from being targeted for degradation by the chaperone Hsp70 and that in the absence of ABL1 activity at least a portion of MLH1 is degraded through the lysosome. This study represents an advance in understanding MMR pathway regulation and has important clinical implications as MMR status is used in the clinic to inform patient treatment, including the use of immunotherapy.

Mutational profiles of head and neck squamous cell carcinomas based upon human papillomavirus status in the Veterans Affairs National Precision Oncology Program.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) associated with human papillomavirus (HPV) demonstrate favorable clinical outcomes compared to patients bearing HPV-negative HNSCC. We sought to characterize the association between HPV status and mutational profiles among patients served by the Veterans Health Administration (VHA). We performed a retrospective analysis of all Veterans with primary HNSCC tumors who underwent next-generation sequencing (NGS) through the VHA's National Precision Oncology Program between July 2016 and February 2019. HPV status was determined by clinical pathology reports of p16 immunohistochemical staining; gene variant pathogenicity was classified using OncoKB, an online precision oncology knowledge database, and mutation frequencies were compared using Fisher's exact test. A total of 79 patients met inclusion criteria, of which 48 (60.8%) had p16-positive tumors. Patients with p16-negative HNSCC were more likely to have mutations in TP53 (p < 0.0001), and a trend towards increased mutation frequency was observed within NOTCH1 (p = 0.032) and within the composite CDK/Rb pathway (p = 0.065). Mutations in KRAS, NRAS, HRAS, and FBXW7 were exclusively identified within p16-positive tumors, and a trend towards increased frequency was observed within the PI3K pathway (p = 0.051). No difference in overall mutational burden was observed between the two groups. In accordance with the previous studies, no clear molecular basis for improved prognosis among patients harboring HPV-positive disease has been elucidated. Though no targeted therapies are approved based upon HPV-status, current efforts to trial PI3K inhibitors and mTOR inhibitors across patients with HPV-positive disease bear genomic rationale based upon the current findings.

Loss of ALK hotspot mutations in relapsed neuroblastoma.

ALK is the most commonly mutated oncogene in neuroblastoma with increased mutation frequency reported at relapse. Here we report the loss of an ALK mutation in two patients at relapse and a paired neuroblastoma cell line at relapse. ALK detection methods including Sanger sequencing, targeted next-generation sequencing and a new ALK Agena MassARRAY technique were used to detect common hotspot ALK variants in tumors at diagnosis and relapse from two high-risk neuroblastoma patients. Copy number analysis including single nucleotide polymorphism array and array comparative genomic hybridization confirmed adequate tumor cell content in DNA used for mutation testing. Case 1 presented with an ALK F1174L mutation at diagnosis with a variant allele frequency (VAF) ranging between 23.5% and 28.5%, but the mutation was undetectable at relapse. Case 2 presented with an ALK R1257Q mutation at diagnosis (VAF=39%-47.4%) which decreased to <0.01% at relapse. Segmental chromosomal aberrations were maintained between diagnosis and relapse confirming sufficient tumor cell content for mutation detection. The diagnostic SKNBE1n cell line harbors an ALK F1174S mutation, which was lost in the relapsed SKNBE2c cell line. To our knowledge, these are the first reported cases of loss of ALK mutations at relapse in neuroblastoma in the absence of ALK inhibitor therapy, reflecting intra-tumoral spatial and temporal heterogeneity. As ALK inhibitors are increasingly used in the treatment of refractory/relapsed neuroblastoma, our study highlights the importance of confirming whether an ALK mutation detected at diagnosis is still present in clones leading to relapse.

Circulating Tumor DNA Identifies Diverse Landscape of Acquired Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Metastatic Colorectal Cancer.

Anti-epidermal growth factor receptor (EGFR) antibodies are effective treatments for metastatic colorectal cancer. Improved understanding of acquired resistance mechanisms may facilitate circulating tumor DNA (ctDNA) monitoring, anti-EGFR rechallenge, and combinatorial strategies to delay resistance. Patients with treatment-refractory metastatic colorectal cancer (n = 169) enrolled on the CO.26 trial had pre-anti-EGFR tissue whole-exome sequencing (WES) compared with baseline and week 8 ctDNA assessments with the GuardantOMNI assay. Acquired alterations were compared between patients with prior anti-EGFR therapy (n = 66) and those without. Anti-EGFR therapy occurred a median of 111 days before ctDNA assessment. ctDNA identified 12 genes with increased mutation frequency after anti-EGFR therapy, including EGFR (P = .0007), KRAS (P = .0017), LRP1B (P = .0046), ZNF217 (P = .0086), MAP2K1 (P = .018), PIK3CG (P = .018), BRAF (P = .048), and NRAS (P = .048). Acquired mutations appeared as multiple concurrent subclonal alterations, with most showing decay over time. Significant increases in copy-gain frequency were noted in 29 genes after anti-EGFR exposure, with notable alterations including EGFR (P < .0001), SMO (P < .0001), BRAF (P < .0001), MET (P = .0002), FLT3 (P = .0002), NOTCH4 (P = .0006), ERBB2 (P = .004), and FGFR1 (P = .006). Copy gains appeared stable without decay 8 weeks later. There were 13 gene fusions noted among 11 patients, all but one of which was associated with prior anti-EGFR therapy. Polyclonal resistance was common with acquisition of ≥ 10 resistance related alterations noted in 21% of patients with previous anti-EGFR therapy compared with 5% in those without (P = .010). Although tumor mutation burden (TMB) did not differ pretreatment (P = .63), anti-EGFR exposure increased TMB (P = .028), whereas lack of anti-EGFR exposure resulted in declining TMB (P = .014). Paired tissue and ctDNA sequencing identified multiple novel mutations, copy gains, and fusions associated with anti-EGFR therapy that frequently co-occur as subclonal alterations in the same patient.

A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Abstract 1516: The role of protein tyrosine kinases in DNA mismatch repair

Abstract The DNA mismatch repair (MMR) pathway and its regulation are necessary for genomic stability. Cells deficient in MMR exhibit increased expansion and contraction of short repeat sequences in the genome, termed microsatellite instability (MSI). Loss of MMR also results in resistance to certain classes of DNA damaging chemotherapeutics. The standard treatment option for most patients includes chemotherapy, which has toxic side effects. Immunotherapy has recently been utilized as a treatment for various cancers. MSI-high cancers respond better to immunotherapy treatment compared to microsatellite stable (MSS) cancers. This increased response is believed to be due to the higher mutation frequencies associated with the MSI-high tumors, which lead to greater neoantigen production. The primary proteins required for MMR are known, however regulation of the pathway is not well understood. The MLH1 protein is critical for MMR and is required for the excision licensing of the daughter DNA strand. Loss or mutation of MLH1 leads to defective MMR, increased mutation frequency, and MSI. Recent studies by our laboratory and others have shown that treatment with a tyrosine kinase inhibitor, Imatinib, leads to decreased MLH1 protein expression, and resistance to MMR-dependent apoptosis. These observations suggest a link between Imatinib-sensitive protein tyrosine kinases (PTKs) and the regulation of MMR. We show that MLH1 protein reduction is not occurring at the mRNA level and is thought to be post-transcriptional. We have identified the Imatinib sensitive target most likely responsible for the MLH1 reduction phenotype. We show that the target protein and MLH1 have a physical interaction that likely prevents degradation targeting of MLH1 by the Hsp70 chaperone. A detailed understanding of MMR regulation by PTKs would significantly advance the knowledge in the field of MMR and have important implications for immunotherapy strategies. Citation Format: Hannah Daniels, Breanna Knicely, Eva M. Goellner. The role of protein tyrosine kinases in DNA mismatch repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1516.

Abstract 1166: Leveraging existing data to identify ancestry-associated features across multiple cancer types

Abstract People with different ancestries inherit different risks and encounter different environmental exposures resulting in different somatic profiles. A lack of knowledge about ancestry-specific alterations is a major barrier to implementing precision medicine, leading to inequities in genetic testing, targeted treatment and clinical trial design for cancer patients from the underserved populations. However, a limited number of cancer cases from non-European populations have been sequenced in research setting, and paired normal samples are often not collected in routine clinical care. Here, we inferred African (AFR), South Asian (SAS), East Asian (EAS), American (AMR) and European (EUR) ancestry from comprehensive genomic profiling (CGP) of over 200,000 tumors and identified 18717, 8588, 6594 patients with significant proportions of AFR, AMR, EAS ancestry, respectively. Using logistic regressions with age of diagnosis, gender and tumor mutation burden (TMB) as covariates, we identified 165 ancestry-associated genes across 14 common cancer types. We used reported race/ethnicity data in the AACR project GENIE cohort for validation. We found that endometrial carcinoma patients with AFR ancestry were enriched with TP53 mutations (OR=2.3, FDR corrected p=5x10-31) but lacked PTEN (OR=0.3, FDR corrected p=2x10-41) and KRAS (OR=0.5, FDR corrected p=8x10-13) mutations. In colorectal cancer, KRAS (OR=1.5, FDR corrected p=4x10-44) and APC (OR=1.4, FDR corrected p=1x10-16) mutations were enriched in patients with AFR ancestry, whereas BRAF mutations were depleted in both AFR (OR=0.5, FDR corrected p=5x10-24) and EAS (OR=0.6, FDR corrected p=6x10-5) patients. CDK12 mutations were enriched in both AFR (OR=1.6, FDR corrected p=2x10-4) and EAS (OR=3.1, FDR corrected p=2x10-7) prostate cancer patients. For patients with AMR ancestry, increased mutation frequency was observed in SETD2 in pancreatic cancer (OR=3.4, FDR corrected p=1x10-4), and VHL in renal clear cell carcinoma (OR=1.9, FDR corrected p=2x10-4). In GBM, TERT alterations were associated with EUR ancestry (OR=1.3, FDR corrected p=0.003). Interestingly, TMB-high status was associated with EUR ancestry in melanoma (OR=5.9, p=6x10-36) and anti-correlated with AFR ancestry in endometrial carcinoma (OR=0.7, p=1x10-6). Our large-scale ancestry analysis characterized the somatic landscape of non-European cancer patients. Future directions include quantifying the germline heritability of identified ancestry-associated mutations using a local ancestry risk score (LRS) and investigating ancestral effects on cancer prognosis and outcome using the GENIE cohort. Citation Format: Jian Carrot-Zhang, Justin Newberg, Garrett Frampton, Rameen Beroukhim. Leveraging existing data to identify ancestry-associated features across multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1166.

Genome-wide protein–DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase

DNA–protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA–protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA–protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

A Collapsed Fingers Subdomain is the Basis for DNA Polymerase β I260M Mutator Activity

DNA Polymerase β fills single nucleotide gaps as a part of the Base Excision Repair pathway (BER); thus, deficiencies in Pol β can lead to increased mutation frequency in the cell, which can result in cancer. Our lab has previously shown that the I260M germline mutation of Pol β, which was first identified in prostate cancer, has reduced nucleotide selectivity in a sequence context dependent manner, incorporating G opposite A. To identify the molecular mechanism of the reduced fidelity of I260M, we studied polymerization using single turnover kinetics, and conformational changes using steady state fluorescence and stopped flow FRET. Our data indicate that the I260M mutation affects the fingers region of Pol β by creating a “collapsed” state in both the opened (in the absence of nucleotide) and closed (prior to chemistry) states. Importantly, we show that the incorrect incoming nucleotide binds more tightly to I260M when compared to the wild‐type by a factor of 3.4x. Based on this data, we found that the collapsed fingers subdomain state of I260M may decrease nucleotide discrimination in I260M, illustrating the importance of the “fingers closing” conformational change for polymerase fidelity. A model is being developed to compare the rate of the “fingers closing” conformational change and the reverse reaction of nucleotide release between I260M and the WT.

Single-cell analysis of somatic mutations in human bronchial epithelial cells in relation to aging and smoking.

Although lung cancer risk among smokers is dependent on smoking dose, it remains unknown if this increased risk reflects an increased rate of somatic mutation accumulation in normal lung cells. Here, we applied single-cell whole-genome sequencing of proximal bronchial basal cells from 33 participants aged between 11 and 86 years with smoking histories varying from never-smoking to 116 pack-years. We found an increase in the frequency of single-nucleotide variants and small insertions and deletions with chronological age in never-smokers, with mutation frequencies significantly elevated among smokers. When plotted against smoking pack-years, mutations followed the linear increase in cancer risk until about 23 pack-years, after which no further increase in mutation frequency was observed, pointing toward individual selection for mutation avoidance. Known lung cancer-defined mutation signatures tracked with both age and smoking. No significant enrichment for somatic mutations in lung cancer driver genes was observed.

Microsatellite mutation frequencies in river otters (Lontra Canadensis) from the Athabasca Oil Sands region are correlated to polycyclic aromatic compound tissue burden.

Mining activities in the Athabasca oil sands region (AOSR) have contributed to an increase of polycyclic aromatic compounds (PACs) locally. However, many PACs found in the AOSR, and the combined effects of PAC mixtures have not been evaluated for genotoxicity in wildlife. Here, we examine whether mutation frequencies in AOSR river otters are correlated to PAC tissue burdens. We used single-molecule polymerase chain reaction (SM-PCR) to measure the mutant frequency of unstable DNA microsatellite loci in the bone marrow of wild river otters (n=11) from the AOSR. Microsatellite mutation frequencies were regressed against liver PAC burden (total, low/high molecular weight [LMW/HMW], and parent/alkylated PACs), and to the distances from where the samples were collected to nearby bitumen upgraders. We found that microsatellite mutation frequency was positively correlated with total liver PAC burden. LMW and alkylated PACs were detected at higher levels and had a stronger positive relationship with mutation frequency than HMW (alkylated and parent) PACs. There were no significant relationships detected between mutation frequency and LMW parent PACs or the distance from bitumen upgraders. Furthermore, pyrogenic and petrogenic signatures suggest PACs in animals with high mutation frequencies were associated with combustion processes; although further investigation is warranted, due to limitations of diagnostic ratio determination with biotic models. Our findings support the hypothesis that PACs found in the AOSR increase mutation frequency in wildlife. Further investigation is required to determine if the elevated PAC levels associated with higher mutation frequency are due to natural exposure or elevated human activity.

The Neoantigen Landscape of the Coding and Noncoding Cancer Genome Space

Tumor mutation burden (TMB) is a measure to predict patient responsiveness to immune checkpoint immunotherapy because with increased mutation frequency, the likelihood of a greater neoantigen burden is increased. Although neoantigen prediction tools exist, tumor neoantigen burden has not been adopted as a measure to predict immunotherapy response. With both measures, current guidelines are limited to the coding regions, but ectopic expression of sequences in the noncoding space may potentially be a source of neoantigens. A pan-cancer cohort of 574 advanced disease stage patients with whole genome and transcriptome sequencing was analyzed to report mutation burden and neoantigen counts within the coding and noncoding regions. The efficacy of tumor neoantigen burden, reported as tumor neoantigen count (TNC), including neoantigens derived from the expression of noncoding regions, compared with TMB as a predictor of response to immunotherapy for 80 patients who had received treatment, was evaluated. TMB was found to be the best predictor of response to immunotherapy, whereas expression-derived TNC from the noncoding regions did not improve prediction of response. Therefore, there is minimal benefit in extending the calculation of TNC to the noncoding space for the purposes of predicting response. However, it is likely that there is a wealth of neoantigens derived from the noncoding space that may impact patient outcomes and treatments.

Single-cell analysis of somatic mutation burden in mammary epithelial cells of pathogenic BRCA1/2 mutation carriers.

Inherited germline mutations in the breast cancer gene 1 (BRCA1) or BRCA2 genes (herein BRCA1/2) greatly increase the risk of breast and ovarian cancer, presumably by elevating somatic mutational errors as a consequence of deficient DNA repair. However, this has never been directly demonstrated by a comprehensive analysis of the somatic mutational landscape of primary, noncancer, mammary epithelial cells of women diagnosed with pathogenic BRCA1/2 germline mutations. Here, we used an accurate, single-cell whole-genome sequencing approach to first show that telomerized primary mammary epithelial cells heterozygous for a highly penetrant BRCA1 variant displayed a robustly elevated mutation frequency as compared with their isogenic control cells. We then demonstrated a small but statistically significant increase in mutation frequency in mammary epithelial cells isolated from the breast of BRCA1/2 mutation carriers as compared with those obtained from age-matched controls with no genetically increased risk for breast cancer.

The Mutation of BTG2 Gene Predicts a Poor Outcome in Primary Testicular Diffuse Large B-Cell Lymphoma.

IntroductionPrimary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a rare and aggressive form of mature B-cell lymphoma commonly found in elder males, but its genetic features are poorly understood. In this study, we had performed target-sequencing of 360 lymphoma-related genes on 76 PT-DLBCL patients with a median age of 65 (33–89). Our data provide a comprehensive understanding of the landscape of mutations in a small subset of PT-DLBCL.MethodsA total of 76 PT-DLBCL patients were sequenced, and their clinical data and follow-up data were collected. The relationship between mutated genes, clinical data and prognosis and survival of PT-DLBCL patients was retrospectively analyzed by statistical software.ResultsWe observed a median of 15 protein-altering variants per patient in our data and was identified recurrent oncogenic mutations of 360 lymphoma-related genes involved in PT-DLBCL, including PIM1 (74%), MYD88 (50%), KMT2D (38%), KMT2C (34%), BTG2 (34%), TBL1XR1 (34%) and ETV6 (24%). Compared with classic DLBCL, PT-DLBCL showed an increased mutation frequency of PIM1, MYD88, BTG2, while NOTCH1 appeared exclusive mutated with PIM1, MSH3 and ETV6. Cox risk model regression analysis showed that age ≥60 years, IPI 3–5 points, BTG2 gene mutation and extranodal organ invasion suggested poor prognosis. Finally, we constructed an OS predict model of PT-DLBCL patients using above factors with a high accuracy.ConclusionIn conclusion, our results revealed genomic characterization of PT-DLBCL, and the mutation of BTG2 was an independent factor predicting a poor prognosis.

The PB1 gene from H9N2 avian influenza virus showed high compatibility and increased mutation rate after reassorting with a human H1N1 influenza virus

BackgroundReassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes.MethodsThe polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS).ResultsWe discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice.ConclusionsThe higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.

Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct.

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species’ triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.

Dynamic changes in genetic diversity, drug resistance mutations, and treatment outcomes of falciparum malaria from the low-transmission to the pre-elimination phase on the islands of S\xe3o Tom\xe9 and Pr\xedncipe

BackgroundWith effective vector control and case management, substantial progress has been made towards eliminating malaria on the islands of São Tomé and Príncipe (STP). This study assessed the dynamic changes in the genetic diversity of Plasmodium falciparum, the anti-malarial drug resistance mutations, and malaria treatment outcomes between 2010 and 2016 to provide insights for the prevention of malaria rebounding.MethodsPolymorphic regions of merozoite surface proteins 1 and 2 (msp1 and msp2) were sequenced in 118 dried blood spots (DBSs) collected from malaria patients who had visited the Central Hospital in 2010–2016. Mutations in the multi-drug resistance I (pfmdr1), chloroquine resistance transporter (pfcrt), and kelch 13 (pfk13) genes were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and sequencing in 111 DBSs. A total of 7482 cases that completed a 28-day follow-up were evaluated for treatment outcomes based on the microscopic results. Regression models were used to characterize factors associated with levels of parasite density and treatment failures.ResultsParasite strains in STP showed significant changes during and after the peak incidence in 2012. The prevalent allelic type in msp1 changed from K1 to MAD20, and that in msp2 changed from 3D7/IC to FC27. The dominant alleles of drug-resistance markers were pfmdr1 86Y, 184F, D1246, and pfcrt 76 T (Y-F-D-T, 51.4%). The average parasite density in malaria cases declined threefold from low-transmission (2010–2013) to pre-elimination period (2014–2016). Logistic regression models showed that patients with younger age (OR for age = 0.97–0.98, p < 0.001), higher initial parasite density (log10-transformed, OR = 1.44, p < 0.001), and receiving quinine treatment (compared to artemisinin-based combination therapy, OR = 1.91–1.96, p < 0.001) were more likely to experience treatment failures during follow-up.ConclusionsPlasmodium falciparum in STP had experienced changes in prevalent strains, and increased mutation frequencies in drug-resistance genes from the low-transmission to the pre-elimination settings. Notably, patients with younger age and receiving quinine treatment were more likely to show parasitological treatment failure during follow-up. Therapeutic efficacy should be carefully monitored to inform future treatment policy in STP.

The combined treatment of Molnupiravir and Favipiravir results in a potentiation of antiviral efficacy in a SARS-CoV-2 hamster infection model.

BackgroundFavipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV-2. First efficacy data have been recently reported in COVID-19 patients.MethodsWe here report on the combined antiviral effect of both drugs in a SARS-CoV-2 Syrian hamster infection model. The infected hamsters were treated twice daily with the vehicle (the control group) or a suboptimal dose of each compound or a combination of both compounds.FindingsWhen animals were treated with a combination of suboptimal doses of Molnupiravir and Favipiravir at the time of infection, a marked combined potency at endpoint is observed. Infectious virus titers in the lungs of animals treated with the combination are reduced by ∼5 log10 and infectious virus are no longer detected in the lungs of >60% of treated animals. When start of treatment was delayed with one day a reduction of titers in the lungs of 2.4 log10 was achieved. Moreover, treatment of infected animals nearly completely prevented transmission to co-housed untreated sentinels. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs of treated animals. In the combo-treated hamsters, an increased frequency of C-to-T mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. Interpretation: Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir/Favipiravir in the treatment of COVID-19. Funding: stated in the acknowledgment.