Frequency Of DNA Mutations Research Articles

Abstract 3786: Genomic and transcriptomic characterization of peritoneal, lung and liver metastases of colorectal carcinoma reveals site-specific differences

Abstract Background: Metastasis in colorectal cancer (CRC) is a critical factor in patient outcomes, with 20% of patients affected at the time of diagnosis and up to 50% developing metachronous metastatic disease later on. Despite the growing importance of metastatic sites as independent predictors of overall survival (OS) and the ongoing development of new treatments, there has been limited effort to personalize systemic treatment based on specific metastatic locations. Aim: This study aims to compare the genomic landscape of different metastatic sites in CRC to detect novel diagnostic and therapeutic targets. Methods: DNA whole-genome-sequencing and RNA gene expression data from metastatic CRC samples were retrieved from the Hartwig Medical Foundation (HMF). Single nucleotide variants (SNVs), somatic copy number aberrations (SCNAs) and structural variants (SVs) were reported by HMF with their pipelines. In addition, focal amplifications and deletions were identified using GISTIC2 (v6.15.30). We included 464 microsatellite stable samples derived from liver metastases (n=369), peritoneal metastases (n=58) and lung metastases (n=37). We compared DNA mutation frequencies between tumors from different metastatic sites (two-sided Mann-Whitney tests) and performed differential gene expression and pathway enrichment analysis. Results: Liver metastases had the highest number of focal SCNAs per sample (median 33 vs 17 in peritoneum (p < 0.001); vs 18 in lung (p < 0.001)). Furthermore, we observed 39 site-specific SCNAs in liver, 9 in peritoneal and 15 in lung metastases. Of these, the most significant focal SCNAs were an amplification within 20q13.12 (79%) and deletion within 1p35.3 (64%) in peritoneal metastases and a deletion within 18q21.1 (84%) in lung metastases. Furthermore, peritoneal metastases displayed less genomic instability, featuring lower ploidy (median 2.65 vs 2.94 in liver (p=0.025); vs 3.15 in lung (p=0.025)) and lower aneuploidy scores (median 19 vs 26 in liver (p=0.016); vs 30 in lung (p=0.016)) while having no significant differences in tumor mutational burden or individual SNVs. Analysis of SVs revealed that peritoneal metastases exhibited fewer deletions (≥10kb; 1.3x fold change increase in liver and lung) and fewer duplications (≥1kb; 1.4x fold change increase in liver). Differential gene expression analysis uncovered higher expression of gene sets involved in the apical junction and the TGF-beta-signaling pathway in peritoneal metastases. Conclusion: Specific focal SCNA and differences in genomic instability exhibit an association with preferential organotropism in CRC metastasis and could offer potential targets for the development of new diagnostic and therapeutic approaches. Citation Format: Nerma Crnovrsanin, Soufyan Lakbir, Renske d. Wit, Steven L. Ketelaars, Niels F. Kok, Petur Snaebjornsson, Gerrit A. Meijer, Sanne Abeln, Remond J. Fijneman. Genomic and transcriptomic characterization of peritoneal, lung and liver metastases of colorectal carcinoma reveals site-specific differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3786.

A Comprehensive Genomic Profiling of Myeloid Malignancies Demonstrates Mutational Spectrum of DNA Variants Including FLT3-ITDs, and Gene Fusions

Introduction Myeloid malignancies are a group of clonal hematopoietic disorders including acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). These diseases are heterogenous and genetically complex and the number of genetic alterations associated with them continues to grow. To address the need for comprehensive genetics research we developed the Oncomine TM Myeloid Assay and the Oncomine TM Myeloid Assay GX v2, which both detect genomic alterations associated with myeloid malignancies including 45 DNA genes and >30 fusion driver genes with the ability to detect >700 fusion isoforms. Our panel includes genetic alterations in AML, MPN, and MDS such as FLT3-Internal Tandem Duplications (ITDs), IDH1/2, CEBPA, CALR, and TP53. Methods Here we describe the genomic profiles of 8503 clinical research samples (including AML, MPN, MDS, CML, CMML, or JMML). A total of 4723 samples were run on the Ion GeneStudio TM S5 System and analyzed using the Oncomine TM Myeloid Research workflow on Ion Reporter TM 5.18. A total of 3780 samples were run on the Ion Torrent TM Genexus Software 6.6 and analyzed using the Oncomine TM Myeloid Assay GX v2. Results The turnaround time (the time between starting the run and NGS data report) was 23-25 hours and the hands-on time was around 1 hour, which enabled fast and accurate profiling of myeloid malignancies, and facilitated the timely availability of results. The success rate of the samples was 100%. Frequency of relevant mutations by gene Some genes had a higher rate of genetic mutations. TET2 has the highest percentage of detection (12.6%, 1068 positive sample/8503 total number of samples). Other important genes with the highest percentage of detection included ASXL1 9.3% (787/8503), DNMT3A 7.8% (665/8503), TP53 7.5%, (640/8503), SRSF2 6.9% (587/8503), JAK2 6.6%, (562/8503), RUNX1 5.2% (446/8503), SF3B1 5.0% (423/8503), IDH2 4.0% (337/8503), NRAS 3.8% (318/8503), FLT3 3.4% (292/8503), ABL1 3.2% (273/8503), BCOR 2.7% (229/8503), NPM1 2.4% (201/8503), and IDH1 1.0% (170/8503). Overall, 4797 (56.4%) of samples had a mutation in at least one gene in the panels, and the average number of mutations per samples was 2.3 within these samples. The most co-occurring gene pairs include ASXL1 and TET2, as well as SRSF2 and TET2, both are detected in more than 2% co-occurring gene pairs. Variant allele frequency by gene We observed a substantial difference between the variant allele frequency (VAF) of relevant DNA mutations (SNP + Insertions + Deletions) in different genes. ANKRD26 has a median VAF of 66%, whereas MYD88 has a median VAF of only 7%. (Fig.1) Mutation spectrum of FLT3ITD variants We observed FLT3-ITD in 3% of samples. We observed the VAF of FLT3-ITD variants averaged around 0.32. Interestingly, the length of the FLT3-ITD duplication is multi-modal, with the highest peak at around 34 bp. The maximum duplication length detected is 151 bp. Mutation spectrums of fusions Of the 8480 samples with RNA fusion profiling, 710 were fusion positive (8.4%). ABL1 is the most common fusion driver (432 samples, 5.1%), with 88% of these being BCR- ABL1. Other common fusion drivers include KMT2A (171 samples, 2.0%; 29% are KMT2A-MLLT3 and 26% are KMT2A-MLLT1), MYH11 (126 samples, 1.5%; all are CBFB-MYH11), RARA (117 samples, 1.4%; all are PML-RARA), RUNX1 (95 samples, 1.1%; 56% are RUNX1-RUNX1T1). A total of 17 distinct driver genes, 49 distinct gene pairs and 110 distinct gene isoforms were observed, including rare fusions (for example, ZMYM2-FGFR1, KAT6A-CREBBP, etc) that are not detectable by traditional methods like qPCR (quantitative polymerase chain reaction) or FISH (fluorescence in-situ hybridization). The read count of the fusion positive samples ranges from 21 to 113,000. (Fig.2) Conclusions The Oncomine Myeloid Assay is a fast, accurate, robust, and reproducible solution for comprehensive genomic profiling of myeloid malignancies. We describe the mutational spectrum of DNA variants and RNA fusions in a range of clinical research samples, exploring the VAF, the abundance of mutations in different genes, and the properties of observed gene fusions. (For research use only. Not for use in diagnostic procedures.)

Whole-genome high-fidelity sequencing: A novel approach to detecting and characterization of mutagenicity in vivo.

Direct DNA sequencing can be used for characterizing mutagenicity in simple and complex biological models. Recently we described a method of whole-genome sequencing for detecting mutations in simple models of cultured bacteria, mammalian cells, and nematode. In the current proof-of-concept study, we expand and improve our method for evaluating a more complex mammalian biological model in outbred mice. We detail the method by applying it to a small set of animals treated with a mutagen with known mutagenicity profiles, N-ethyl-N-nitrosourea (ENU), for consistency with the known data. Whole-genome high-fidelity sequencing (HiFi Sequencing) showed frequencies and spectra of background mutations in tissues of untreated mice that were consistent with normal ageing and characterized by spontaneous or enzymatic deamination of 5-methylcytosine. In mice treated with a single 40mg/kg dose of ENU, the frequency of mutations in the genomic DNA of solid tissues increased up to 7-fold, with the greatest increase observed in the spleen and the smallest increase in the liver. The most common mutations detected in ENU-treated mice were T>A transitions and T>C transversions, consistent with the types of mutations caused by alkylating agents. The data suggest that HiFi Sequencing may be useful for characterizing mutagenicity of novel compounds in various biological models.

Effective differentiation of double negative thymocytes requires high fidelity replication of mitochondrial DNA in an age dependent manner.

One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase γ (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA.

Mitochondrial DNA Polymorphism in HV1 and HV2 Regions and 12S rDNA in Perimenopausal Hypertensive Women.

Estrogens enhance cellular mitochondrial activity. The diminution of female hormones during menopause may have an effect on the mitochondrial genome and the expression of mitochondrial proteins. Hence, oxidative stress and the pro-inflammatory state contribute to the formation of systemic illnesses including arterial hypertension (AH). This study aimed to determine the types and frequency of mutations in the mitochondrial DNA (mtDNA) nucleotide sequence in the hypervariable regions 1 and 2 (HV1 and HV2) and the 12S RNA coding sequence of the D-loop in postmenopausal women with hypertension. In our study, 100 women were investigated, 53 of whom were postmenopausal and 47 of whom were premenopausal (53.9 ± 3.7 years vs. 47.7 ± 4.2 years, respectively). Of those studied, 35 premenopausal and 40 postmenopausal women were diagnosed with AH. A medical checkup with 24 h monitoring of blood pressure (RR) and heart rate was undertaken (HR). The polymorphism of the D-loop and 12S rDNA region of mtDNA was examined. Changes in the nucleotide sequence of mtDNA were observed in 23% of the group of 100 women. The changes were identified in 91.3% of HV1 and HV2 regions, 60.9% of HV1 segments, 47.5% of HV2 regions, and 43.5% of 12S rDNA regions. The frequency of nucleotide sequence alterations in mtDNA was substantially higher in postmenopausal women (34%) than in premenopausal women (10.6%), p = 0.016. A higher frequency of changes in HV1 + HV2 sections in postmenopausal women (30.2%) compared to the premenopausal group (10.6%) was detected, p = 0.011. Only postmenopausal women were found to have modifications to the HV2 segment and the 12S rDNA region. After menopause, polymorphism in the mtDNA region was substantially more frequent in women with arterial hypertension than before menopause (p = 0.030; 37.5% vs. 11.5%). Comparable findings were observed in the HV2 and HV1 regions of the AH group (35% vs. 11.5%), p = 0.015, in the HV1 segment (25% vs. 11.5%), p = 0.529, and in the HV2 segment, 12S rDNA (25% vs. 0%). More than 80% of all changes in nucleotide sequence were homoplasmic. The mtDNA polymorphisms of the nucleotide sequence in the HV1 and HV2 regions, the HV2 region alone, and the 12S RNA coding sequence were associated with estrogen deficiency and a more severe course of arterial hypertension, accompanied by symptoms of adrenergic stimulation.

Effect of DPP4/CD26 expression on SARS‑CoV‑2 susceptibility, immune response, adenosine (derivatives m62A and CD) regulations on patients with cancer and healthy individuals.

The worldwide COVID‑19 pandemic was brought on by a new coronavirus (SARS Cov‑2). A marker/receptor called Dipeptidyl peptidase 4/CD26(DPP4/CD26) may be crucial in determining susceptibility to tumors and coronaviruses. However, the regulation of DPP4 in COVID‑invaded cancer patients and its role on small molecule compounds remain unclear. The present study used the Human Protein Atlas, Monaco, and Schmiedel databases to analyze the expression of DPP4 in human tissues and immune cells. The association between DPP4 expression and survival in various tumor tissues was compared using GEPIA 2. The DNMIVD database was used to analyze the correlation between DPP4 expression and promoter methylation in various tumors. On the cBioPortal network, the frequency of DPP4 DNA mutations in various cancers was analyzed. The correlation between DPP4 expression and immunomodulators was analyzed by TISIDB database. The inhibitory effects of cordycepin (CD), N6, N6‑dimethyladenosine (m62A) and adenosine (AD) on DPP4 in cancer cells were evaluated. DPP4 was mainly expressed in endocrine tissue, followed by gastrointestinal tract, female tissue (mainly in placenta), male tissue (mainly in prostate and seminal vesicle), proximal digestive tract, kidney, bladder, liver, gallbladder and respiratory system. In immune cells, DPP4 mRNA was mainly expressed in T cells, and its expression was upregulated in esophageal carcinoma, kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, stomach adenocarcinoma, thyroid carcinoma and thymoma. However, it was downregulated in breast invasive carcinoma, kidney chromophobe, lung squamous cell carcinoma and skin cutaneous melanoma. Thus, DPP4 is involved in viral invasion in most types of cancer. The expression of DPP4 could be inhibited by CD, m62A and AD in different tumor cells. Moreover, CD significantly inhibited the formation of GFP‑positive syncytial cells. Invivo experiments with AD injection further showed that AD significantly inhibited lymphocyte activating factor 3 expression. These drugs may have potential to treat COVID‑19 by targeting DPP4. Thus, DPP4 may be medically significant for SARS‑CoV‑2‑infected cancer patients, providing prospective novel targets and concepts for the creation of drugs against COVID‑19.

A pilot study to evaluate tissue- and plasma-based DNA driver mutations in a cohort of patients with pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions to pancreatic ductal adenocarcinoma that are challenging to manage due to limited imaging, cytologic, and molecular markers that accurately classify lesions, grade of dysplasia, or focus of invasion preoperatively. The objective of this pilot study was to determine the frequency and type of DNA mutations in a cohort of surgically resected, pathologically confirmed IPMN, and to determine if concordant mutations are detectable in paired pretreatment plasma samples. Formalin-fixed paraffin-embedded (FFPE) tissue from 46 surgically resected IPMNs (31 low-grade, 15 high-grade) and paired plasma from a subset of 15 IPMN cases (10 low-grade, 5 high-grade) were subjected to targeted mutation analysis using a QIAseq Targeted DNA Custom Panel. Common driver mutations were detected in FFPE from 44 of 46 (95.6%) IPMN cases spanning all grades; the most common DNA mutations included: KRAS (80%), RNF43 (24%), and GNAS (43%). Of note, we observed a significant increase in the frequency of RNF43 mutations from low-grade to high-grade IPMNs associated or concomitant with invasive carcinoma (trend test, P = 0.01). Among the subset of cases with paired plasma, driver mutations identified in the IPMNs were not detected in circulation. Overall, our results indicate that mutational burden for IPMNs is a common occurrence, even in low-grade IPMNs. Furthermore, although blood-based biopsies are an attractive, noninvasive method for detecting somatic DNA mutations, the QIAseq panel was not sensitive enough to detect driver mutations that existed in IPMN tissue using paired plasma in the volume we were able to retrieve for this retrospective study.

Abstract 1260: Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes

Abstract Background: Rucaparib is a PARP inhibitor approved to treat ovarian cancer (OC) and metastatic castration-resistant prostate cancer (mCRPC). Deficiencies in HRR resulting from genetic or epigenetic alterations in BRCA1/2 sensitize tumors to rucaparib through synthetic lethality. Inactivation of other HRR genes may also confer sensitivity to PARP inhibition. Here, we evaluated rucaparib efficacy in preclinical models with genomic or epigenetic alterations in an HRR gene panel. Methods: BRCA1/2 and 14 additional genes were selected for their role in DNA repair and mutation frequency in OC and mCRPC. Small interfering RNA (siRNA) knockdowns were performed in OC (OVCAR-3, OAW28, SK-OV-3) and mCRPC (DU145, PC-3) cell lines to model their inactivation and assess impact on rucaparib sensitivity (IC50) in cell viability assays. Patient-derived xenografts (PDX) harboring deleterious alterations in BRCA1/2, BARD1, BRIP1, NBN, PALB2, RAD51B, RAD51C, or RAD51D, or BRCA1/RAD51C promoter hypermethylation were treated with up to 150 mg/kg rucaparib BID for 25-42 days and tumor growth was monitored. Mutational status was confirmed by next-generation sequencing. Results: In addition to BRCA1/2 knockdown, depletion of BARD1, PALB2, or RAD51 resulted in potent rucaparib activity in OC and mCRPC cells, where the rucaparib IC50 was reduced by >70% vs control in at least 4 of the 5 cell lines tested. Rucaparib also displayed greater potency in cells with siRNA-mediated decreased expression of FANCA, NBN, RAD51C, or RAD54L, where at least 1 cell line had >50% decrease in IC50. Rucaparib treatment in 19/33 HRR-deficient PDX models resulted in significant tumor growth inhibition (TGI). These included BRCA1/2, PALB2, NBN, RAD51B, RAD51C, or RAD51D-mutated and BRCA1/RAD51C-hypermethylated models. Efficacy was strongly associated with biallelic inactivation of BRCA1/2, PALB2, RAD51C, or RAD51D with 87% vs 29% mean TGI for monoallelic BRCA1/2 alterations (P<0.0001). In addition, a single PDX model each for the exploratory genes NBN and RAD51B had 101% and 80% TGI, respectively. No significant difference was observed between PDX models with BRCA1/2 vs non-BRCA1/2 (PALB2, NBN, RAD51B, RAD51C, or RAD51D) biallelic alterations (98% vs 80% mean TGI, respectively; P=0.21). In support of these nonclinical findings, we highlight a patient with RAD51B-mutant mCRPC treated with rucaparib in the TRITON2 trial (NCT02952534) who had confirmed radiographic and PSA responses. Conclusions: Cell lines and PDX models with biallelic alterations in HRR genes other than BRCA1/2, including RAD51B, RAD51C, RAD51D, PALB2, and NBN, are comparably sensitive to rucaparib. Rucaparib is being evaluated in the LODESTAR trial (NCT04171700) in patients with tumors associated with deleterious alterations in BRCA1/2, PALB2, RAD51C, and RAD51D (cohort A) and BARD1, BRIP1, FANCA, NBN, RAD51, and RAD51B (exploratory cohort B). Citation Format: Liliane Robillard, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, Minh Nguyen. Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes [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 1260.

The radiation adaptive response and priming dose influence: the quantification of the Raper\u2013Yonezawa effect and its three-parameter model for postradiation DNA lesions and mutations

The priming dose effect, called also the Raper–Yonezawa effect or simply the Yonezawa effect, is a special case of the radiation adaptive response phenomenon (radioadaptation), which refers to: (a) faster repair of direct DNA lesions (damage), and (b) DNA mutation frequency reduction after irradiation, by applying a small priming (conditioning) dose prior to the high detrimental (challenging) one. This effect is observed in many (but not all) radiobiological experiments which present the reduction of lesion, mutation or even mortality frequency of the irradiated cells or species. Additionally, the multi-parameter model created by Dr. Yonezawa and collaborators tried to explain it theoretically based on experimental data on the mortality of mice with chronic internal irradiation. The presented paper proposes a new theoretical approach to understanding and explaining the priming dose effect: it starts from the radiation adaptive response theory and moves to the three-parameter model, separately for two previously mentioned situations: creation of fast (lesions) and delayed damage (mutations). The proposed biophysical model was applied to experimental data—lesions in human lymphocytes and chromosomal inversions in mice—and was shown to be able to predict the Yonezawa effect for future investigations. It was also found that the strongest radioadaptation is correlated with the weakest cellular radiosensitivity. Additional discussions were focussed on more general situations where many small priming doses are used.

Uterine lavage for the detection of ovarian cancer using an expanded gene panel

Uterine lavage for the detection of ovarian cancer using an expanded gene panel

Abstract LB047: Uterine lavage for the detection of ovarian cancer using an expanded gene panel

Abstract Objectives: Current screening methods for ovarian cancer have failed to demonstrate a significant reduction in mortality. Uterine lavage catheters have been used to detect tumor-specific TP53 mutations from cells presumably shed from high-grade serous ovarian cancer, but this technique may not identify non-serous subtypes or early-stage disease. We aimed to pilot the combination of deep sequencing methods with an expanded gene panel to improve detection of both early stage and non-serous ovarian cancers. Methods: Lavage of the uterine cavity was performed in 35 consecutive patients undergoing surgery with preoperative concern for an ovarian malignancy. Duplex sequencing, an ultra-accurate error-correction sequencing approach, was used to deeply sequence extracted DNA from lavage samples (average duplex depth ~2500x) with a panel of candidate ovarian cancer driver genes including TP53, ARID1A, PTEN, PPP2R1A, CDKN2A, KRAS (whole genes), CTNNB1, PIK3CA, and BRAF (hotspots only). Tumor DNA was sequenced to identify driver mutations and compare with mutations found in the lavages. The overall mutation frequency in lavage DNA was calculated by dividing identified non-polymorphism mutant alleles by the total number of nucleotides sequenced in coding regions. Results: In total, lavage samples were collected from fourteen women with benign disease, thirteen with high grade serous carcinoma (HGSC), three with clear cell carcinoma, three with endometrioid carcinoma, one with granulosa cell carcinoma and one with carcinosarcoma. Thirteen women had stage I or II disease (including five with stage I or II high grade serous). Processed lavage samples yielded a median DNA of 596.5 ng. Tumor sequencing is ongoing, but of seven fully sequenced lavage/tumor pairs, the tumor-specific mutation was identified in four lavage samples: TP53 mutations found in two HGSC (stage IIb and stage III), an ARID1A mutation from a stage Ic3 clear cell carcinoma, and a PIK3CA mutation from a stage Ia endometrioid carcinoma. The tumor-specific mutation was not identified in lavage samples from two patients with endometrioid and one with clear cell carcinoma. Of 21 lavage samples that have presently undergone duplex sequencing, a total of 596 additional somatic mutations were identified in the nine genes. Lavages from patients with HGSC tended to have increased average mutation frequency of TP53 compared to patients with benign disease (TP53 2.2x10-6 vs 9.2x10-7, p=0.09). Conclusion: Sequencing at high depth and using an expanded gene panel allows for the identification of tumor mutations in uterine lavage samples, including some early stage and non-serous ovarian cancers, as well as identifying significant somatic mutational background. Larger studies are needed to confirm the clinical utility of this method, which may have potential to improve the early detection of ovarian cancer. Citation Format: Talayeh Ghezelayagh, Jeanne Fredrickson, Enna Manhardt, Marc R. Radke, Brendan Kohrn, Heidi J. Gray, Renata R. Urban, Kathryn P. Pennington, John B. Liao, Kemi M. Doll, Elise J. Simons, Jennifer K. Burzawa, Barbara A. Goff, Paul Speiser, Elizabeth M. Swisher, Rosa Ana Risques, Barbara M. Norquist. Uterine lavage for the detection of ovarian cancer using an expanded gene panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB047.

Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence

The development of complex phenotypes in industrially relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed toward increasing mutation frequencies, yet the precise control of mutagenesis processes in cell factories continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in the bacterial domain. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in DNA mutation frequencies and a heritable fixation of mutations in the genome. Following such an accelerated mutagenesis-followed by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin (either individually or combined) and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate the evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes toward the desired phenotype(s).

Mitochondrial gene mutations in pediatric septic shock.

There has been a growing interest in the association between mitochondrial dysfunction and sepsis. However, most studies have focused on mitochondrial structural damage, functional aspects, or the clinical phenotypes in sepsis. The purpose of this study was to evaluate mitochondrial DNA (mtDNA) gene mutations in critically ill pediatric patients with septic shock. Thirteen patients with severe sepsis or septic shock admitted to the pediatric intensive care unit (PICU) of a tertiary children's hospital were enrolled in this prospective observational study. Clinical data from electronic medical records were obtained. Whole-blood samples were collected within 24 h of PICU admission to perform PBMC isolation, mtDNA extraction, and mtDNA sequencing using next-generation sequencing. mtDNA sequencing revealed mutations in 9 of the 13 patients, presenting 27 point mutations overall, with 15 (55.6%) located in the locus related to adenosine triphosphate production and superoxide metabolism, including electron transport. In this pilot study, significant numbers of mtDNA point mutations were detected in critically ill pediatric patients with septic shock. These mutations could provide promising evidence for mitochondrial dysfunction in sepsis and a basis for further large-scale studies. This study is the first to examine mitochondrial DNA mutations in pediatric patients with septic shock using next-generation sequencing. A high frequency of mitochondrial DNA mutations was detected in these patients indicating an association with septic shock. This pilot study may provide a potential explanation for the association between mitochondrial dysfunction and septic shock on a genetic basis.

Genotype-by-Sequencing Analysis of Mutations and Recombination in Pepper Progeny of Gamma-Irradiated Gametophytes.

The irradiation of dry seeds is the most widely-used irradiation method for improving seed-propagated crops; however, the irradiation of other tissues also has useful effects. The irradiation of plant reproductive organs, rather than seeds, for mutation breeding has advantages, such as producing non-chimeric progeny. However, the mutation frequency and spectrum produced using this method have not been analyzed on a genome-wide level. We performed a genotype-by-sequencing analysis to determine the frequencies of single-base substitutions and small (1–2 bp) insertions and deletions in hot pepper (Capsicum annuum L.) plants derived from crosses using gamma-irradiated female or male gametophytes. The progeny of irradiated gametophytes showed similar or higher DNA mutation frequencies, which were dependent on the irradiation dose and irradiated tissue, and less biased single base substitutions than progeny of irradiated seeds. These characteristics were expected to be beneficial for development of mutation population with a high frequency of small DNA mutations and performing reverse-genetics-based mutation screening. We also examined the possible use of this irradiation method in manipulating the meiotic recombination frequency; however, no statistically significant increase was detected. Our results provide useful information for further research and breeding using irradiated gametophytes.

Molecular Barcode Sequencing for Highly Sensitive Detection of Circulating Tumor DNA in Patients with Esophageal Squamous Cell Carcinoma

Introduction:Next-generation sequencing (NGS) with molecular barcodes (MB) is a novel method that enables the highly sensitive detection of circulating tumor DNA (ctDNA) in a relatively wide range of genes. Objective:The aim of this study was to examine the utility of NGS with MB for detecting ctDNA in patients with esophageal squamous cell carcinoma (ESCC). Methods:Five patients with ESCC who underwent preoperative treatment followed by esophagectomy were examined. The frequency of TP53 mutations in DNA extracted from tumor tissue and plasma at each time point during the treatment course was analyzed using NGS without MB. In 1 patient, additional analysis using NGS with MB was conducted to compare the sensitivities and to evaluate the clinical utility of this novel method. Results:TP53 mutations in tumor tissue were identified in 3 of 5 patients with ESCC. In 1 patient, the mutational allele frequency in plasma was 1.97% before preoperative treatment, and decreased to 0.09% after preoperative treatment. As the maximum frequency of background errors were 3.22% using NGS without MB and 0.08% with MB, which indicated that the sensitivity of ctDNA detection using NGS with MB was much higher than without MB. In 1 patient who had recurrence half a year after surgery, only NGS with MB could detect ctDNA even at 4 weeks after surgery, at a frequency of 0.20%. Conclusions:NGS with MB enabled comprehensive and highly sensitive detection of ctDNA in a patient with ESCC. This novel method may be useful for the clinical diagnosis of ESCC.

Mitochondrial biology in airway pathogenesis and the role of NRF2

A constant improvement in understanding of mitochondrial biology has provided new insights into mitochondrial dysfunction in human disease pathogenesis. Impaired mitochondrial dynamics caused by various stressors are characterized by structural abnormalities and leakage, compromised turnover, andreactive oxygen species overproduction in mitochondria as well as increased mitochondrial DNA mutation frequency, which leads to modified energy production and mitochondria-derived cell signaling. The mitochondrial dysfunction in airway epithelial, smooth muscle, and endothelial cells has been implicated in diseases including chronic obstructive lung diseases and acute lung injury. Increasing evidence indicates that the NRF2-antioxidant response element (ARE) pathway not only enhances redox defense but also facilitates mitochondrial homeostasis and bioenergetics. Identification of functional or potential AREs further supports the role for Nrf2 in mitochondrial dysfunction-associated airway disorders. While clinical reports indicate mixed efficacy, NRF2 agonists acting on respiratory mitochondrial dynamics are potentially beneficial. In lung cancer, growth advantage provided by sustained NRF2 activation is suggested to be through increased cellular antioxidant defense as well as mitochondria reinforcement and metabolic reprogramming to the preferred pathways to meet the increased energy demands of uncontrolled cell proliferation. Further studies are warranted to better understand NRF2 regulation of mitochondrial functions as therapeutic targets in airway disorders.

Phase 1 trial of dasatinib combined with afatinib for epidermal growth factor receptor- (EGFR-) mutated lung cancer with acquired tyrosine kinase inhibitor (TKI) resistance

BackgroundBypass activation of Src family kinases can confer resistance to EGFR tyrosine kinase inhibitors (TKIs) based on preclinical models. We prospectively assessed the safety and clinical activity of dasatinib and afatinib in combination for patients with resistant EGFR-mutant lung cancer.MethodsAn open-label, dose-escalation phase 1/2 trial (NCT01999985) with 2-stage expansion was conducted with 25 lung cancer patients. Dose expansion required activating EGFR mutations and progression following prior EGFR TKI.ResultsPatients were 72% Caucasian and received median of 2 prior lines of therapy. Maximum-tolerated dose was 30 mg afatinib with 100 mg dasatinib. New or increased pleural effusions were observed in 56% of patients. No radiologic responses were observed, although several EGFR-mutant TKI-resistant patients (26%) had prolonged stable disease over 6 months. The combination reduced the EGFR mutation and T790M variant allele frequency in cell-free DNA (p < .05). Nonetheless, the threshold for futility was met, based on 6-month progression-free survival. For EGFR TKI-resistant patients, median progression-free survival was 3.7 months (95% confidence interval (CI), 2.3–5.0) and overall survival was 14.7 months (95% CI, 8.5–20.9).ConclusionsThe combination had a manageable toxicity profile and in vivo T790M modulation, but no objective clinical responses were observed.

Outcome and molecular characteristics of non-invasive encapsulated follicular variant of papillary thyroid carcinoma with oncocytic features.

In 2016, non-invasive encapsulated follicular variant of papillary thyroid carcinoma (NI-EFVPTC) was renamed as noninvasive thyroid follicular neoplasm with papillary-like nuclear features (NIFTP). However, as the study cohort did not mention tumors with oncocytic features, such lesions are still labeled by some as FVPTC. It is therefore crucial to evaluate the outcome and molecular profile of oncocytic NI-EFVPTC. A multi-institutional clinico-pathologic review was conducted to select 61 patients having oncocytic NI-EFVPTC. A detailed molecular profile was carried out in 15 patients. Oncocytic NI-EFVPTCs predominantly affected women in their 50s. There was no distant metastasis, lymph node metastases, or structural recurrence in the entire cohort. Among patients with ≥5 years of FU, all 33 individuals did not recur with a median FU of 10.2 years. Oncocytic NI-EFVPTC commonly had RAS (33%) mutations, a high frequency of mitochondrial DNA mutations (67%) and multiple chromosomal gains/losses (53%). No fusion genes were detected. Oncocytic NI-EFVPTC, when stringently selected for, lacks metastasis at presentation and follows an extremely indolent clinical course, even when treated conservatively with lobectomy alone without RAI therapy. These tumors share a similar mutational profile as NIFTP, FVPTC, and follicular neoplasm and are predominantly RAS-related. Like Hurthle cell neoplasms, they harbor a high frequency of mitochondrial DNA mutations, which contribute to the oncocytic cytomorphology. However, they lack the widespread chromosomal alterations observed in Hurthle cell carcinoma. Consideration should be given to include oncocytic NI-EFVPTCs as NIFTP in order to avoid overtreatment of these highly indolent tumors.

PO-002 Angiotensin II-induced hypertension increases the mutation frequency in the rat kidney

IntroductionEpidemiological studies revealed an increased risk for kidney cancer in hypertensive patients. In many of these patients, the blood pressure regulating renin angiotensin aldosterone system (RAAS) is activated. A stimulated RAAS can lead to oxidative stress and DNA damage, as we have shown both in vitro and in animal models of hypertension. Here, we used a rat model to quantify mutations generated by 20 weeks of angiotensin II-infusion.Material and methodsBigBlue+/- rats, which carry a transgenic lacI gene for mutation analysis, were treated with 0.4 mg angiotensin II/kg/day with the help of osmotic minipumps. Urinary samples were collected in week 15 by placing the rats into metabolic cages. After 20 weeks of treatment the kidneys were isolated and analysed for oxidative stress markers, DNA damage and mutation frequency.Results and discussionsAngiotensin II-treated animals showed significantly increased levels of blood pressure. Urinary and blood parameters of oxidative stress to proteins and nucleobases were increased. Lipoxygenation products were reduced in urine compared to the control animals, pointing to impaired renal excretion of these substances. Oxidative stress and the surrogate marker for DNA double-strand breaks, γ-H2AX, was significantly increased in the renal cortex. Further, a significant elevation of the mutation frequency in kidney DNA was detected. Sequencing results revealed the presence of G->T transversions in the mutated reporter gene of the angiotensin II-treated animals while no such transversions were found in the mutated reporter gene from the control animals. G->T transversions are mutations arising typically from the unrepaired oxidised base modification 8-oxodG.ConclusionThis study shows evidence that the oxidative stress and DNA damage previously observed in vitro and in animal models of hypertension after angiotensin II treatment is indeed associated with the formation of mutations in rat kidneys, providing further evidence for a cancer initiating potential of increased angiotensin II concentrations.

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.