Low-frequency Mutations Research Articles

Impact of genomic alterations measured in circulating tumor DNA (ctDNA) on clinical response to telisotuzumab vedotin treatment in patients with non-small cell lung cancer (NSCLC).

9032 Background: The antibody-drug conjugate telisotuzumab vedotin (Teliso-V) is composed of the c-Met–targeting antibody telisotuzumab (ABT-700) linked to the microtubule inhibitor monomethyl auristatin E. In the LUMINOSITY study (NCT03539536), efficacy of Teliso-V was seen in patients (pts) with EGFR wildtype nonsquamous NSCLC and c-Met overexpression (≥25% tumor cells at 3+ intensity by IHC); overall response rate: 36.5%. Data are limited on whether specific driver oncogene states affect responses. We investigated genomic alterations in relation to response to Teliso-V in this population. Methods: Pts received 1.9 mg/kg Teliso-V monotherapy once every 2 weeks in LUMINOSITY. ctDNA was isolated from plasma collected at different timepoints. The PGDx elio plasma complete assay was used to identify genomic alterations in ctDNA samples. This assay included 521 genes for single nucleotide variants and insertion-deletion mutations, 38 for amplifications (amp), and 21 for translocations. Results: In total, 52 pts were included in the study; ctDNA from 48 pts was analyzed. The overall response rate among pts with ctDNA results was 37.5% (18 pts with partial response) compared with 36.5% for the ITT population of 52 pts. Genomic alterations are listed in the Table.Three out of 4 pts with MET amp at baseline responded, accounting for 17% of total responses. The observed MET amp frequency in this MET IHC preselected cohort was 8%, which is similar to the prevalence observed in tissue analysis by FISH. Of note, 1 nonresponder harbored a MET ex14del mutation at baseline, and a responder had a low-frequency mutation detected at the final visit. Mutations in KRAS were the most common genomic alteration and were detected in 13 (27%) pts at baseline. Three pts with a KRAS mutation were responders; among these, 2 out of 3 had a KRAS G12C mutation (seen in 3 pts total). Response rates were higher in pts with MET amp (75%; 95% CI: 0.30, 0.95) vs those without MET amp (34%; 0.22, 0.49), and higher in pts without KRAS mutations (43%; 0.28, 0.59) vs those with KRAS mutations (23%; 0.08, 0.50); however, confidence intervals were wide and larger sample sizes are needed. Conclusions: MET amp occurred more frequently in responders; however, Teliso-V activity was not restricted to these pts, as most responders were not MET amplified. Specific genomic alterations beyond MET may influence clinical response. The current analysis demonstrated numeric differences between pts with identified drivers who did or did not respond to Teliso-V. Additional research on this topic is needed in larger pt cohorts and/or with tissue-based NGS analyses. [Table: see text]

Comparison and Characterization of Phenotypic and Genomic Mutations Induced by a Carbon-Ion Beam and Gamma-ray Irradiation in Soybean (Glycine max (L.) Merr.).

Soybean (Glycine max (L.) Merr.) is a nutritious crop that can provide both oil and protein. A variety of mutagenesis methods have been proposed to obtain better soybean germplasm resources. Among the different types of physical mutagens, carbon-ion beams are considered to be highly efficient with high linear energy transfer (LET), and gamma rays have also been widely used for mutation breeding. However, systematic knowledge of the mutagenic effects of these two mutagens during development and on phenotypic and genomic mutations has not yet been elucidated in soybean. To this end, dry seeds of Williams 82 soybean were irradiated with a carbon-ion beam and gamma rays. The biological effects of the M1 generation included changes in survival rate, yield and fertility. Compared with gamma rays, the relative biological effectiveness (RBE) of the carbon-ion beams was between 2.5 and 3.0. Furthermore, the optimal dose for soybean was determined to be 101 Gy to 115 Gy when using the carbon-ion beam, and it was 263 Gy to 343 Gy when using gamma rays. A total of 325 screened mutant families were detected from out of 2000 M2 families using the carbon-ion beam, and 336 screened mutant families were found using gamma rays. Regarding the screened phenotypic M2 mutations, the proportion of low-frequency phenotypic mutations was 23.4% when using a carbon ion beam, and the proportion was 9.8% when using gamma rays. Low-frequency phenotypic mutations were easily obtained with the carbon-ion beam. After screening the mutations from the M2 generation, their stability was verified, and the genome mutation spectrum of M3 was systemically profiled. A variety of mutations, including single-base substitutions (SBSs), insertion-deletion mutations (INDELs), multinucleotide variants (MNVs) and structural variants (SVs) were detected with both carbon-ion beam irradiation and gamma-ray irradiation. Overall, 1988 homozygous mutations and 9695 homozygous + heterozygous genotype mutations were detected when using the carbon-ion beam. Additionally, 5279 homozygous mutations and 14,243 homozygous + heterozygous genotype mutations were detected when using gamma rays. The carbon-ion beam, which resulted in low levels of background mutations, has the potential to alleviate the problems caused by linkage drag in soybean mutation breeding. Regarding the genomic mutations, when using the carbon-ion beam, the proportion of homozygous-genotype SVs was 0.45%, and that of homozygous + heterozygous-genotype SVs was 6.27%; meanwhile, the proportions were 0.04% and 4.04% when using gamma rays. A higher proportion of SVs were detected when using the carbon ion beam. The gene effects of missense mutations were greater under carbon-ion beam irradiation, and the gene effects of nonsense mutations were greater under gamma-ray irradiation, which meant that the changes in the amino acid sequences were different between the carbon-ion beam and gamma rays. Taken together, our results demonstrate that both carbon-ion beam and gamma rays are effective techniques for rapid mutation breeding in soybean. If one would like to obtain mutations with a low-frequency phenotype, low levels of background genomic mutations and mutations with a higher proportion of SVs, carbon-ion beams are the best choice.

Discovery and biological evaluation of an adamantyl-amide derivative with likely MmpL3 inhibitory activity

A series of molecules containing bulky lipophilic scaffolds was screened for activity against Mycobacterium tuberculosis and a number of compounds with antimycobacterial activity were identified. The most active compound, (2E)-N-(adamantan-1-yl)-3-phenylprop-2-enamide (C1), has a low micromolar minimum inhibitory concentration, low cytotoxicity (therapeutic index = 32.26), low mutation frequency and is active against intracellular Mycobacterium tuberculosis. Whole genome sequencing of mutants resistant to C1 showed a mutation in mmpL3 which may point to the involvement of MmpL3 in the antimycobacterial activity of the compound. In silico mutagenesis and molecular modelling studies were performed to better understand the binding of C1 within MmpL3 and the role that the specific mutation may play in the interaction at protein level. These analyses revealed that the mutation increases the energy required for binding of C1 within the protein translocation channel of MmpL3. The mutation also decreases the solvation energy of the protein, suggesting that the mutant protein might be more solvent-accessible, thereby restricting its interaction with other molecules. The results reported here describe a new molecule that may interact with the MmpL3 protein, providing insights into the effect of mutations on protein-ligand interactions and enhancing our understanding of this essential protein as a priority drug target.

Low frequency of AIP mutations in patients with young-onset sporadic pituitary macroadenomas

PurposeMutations in the aryl hydrocarbon receptor interacting protein (AIP) gene cause familial isolated pituitary adenomas (FIPA). AIP mutations have also been found in patients with apparently sporadic pituitary adenomas, particularly in young patients with large adenomas. The aim of this study was to determine the frequency of AIP germline mutations in patients with young-onset sporadic pituitary macroadenomas.MethodsThe AIP gene was sequenced in 218 Portuguese patients with sporadic pituitary macroadenomas diagnosed before the age of 40 years.ResultsHeterozygous rare sequence variants in AIP were identified in 18 (8.3%) patients. However, only four (1.8%) patients had pathogenic or likely pathogenic variants. These consisted of two already known mutations (p.Arg81* and p.Leu115Trpfs*41) and two novel mutations (p.Glu246*, p.Ser53Thrfs*36). All four patients had GH-secreting adenomas diagnosed between the ages of 14 and 25 years. The frequency of AIP pathogenic or likely pathogenic variants in patients under the age of 30 and 18 years was 3.4% and 5.0%, respectively.ConclusionThe frequency of AIP mutations in this cohort was lower than in other studies. Previous reports may have overestimated the contribution of AIP mutations due to the inclusion of genetic variants of uncertain significance. The identification of novel AIP mutations expands the known spectrum of genetic causes of pituitary adenomas and may help understand the role of AIP mutations in the molecular mechanisms underlying pituitary tumorigenesis.

Extensive intratumor regional epigenetic heterogeneity in clear cell renal cell carcinoma targets kidney enhancers and is associated with poor outcome

BackgroundClear cell renal cell cancer (ccRCC), the 8th leading cause of cancer-related death in the US, is challenging to treat due to high level intratumoral heterogeneity (ITH) and the paucity of druggable driver mutations. CcRCC is unusual for its high frequency of epigenetic regulator mutations, such as the SETD2 histone H3 lysine 36 trimethylase (H3K36me3), and low frequency of traditional cancer driver mutations. In this work, we examined epigenetic level ITH and defined its relationships with pathologic features, aspects of tumor biology, and SETD2 mutations.ResultsA multi-region sampling approach coupled with EPIC DNA methylation arrays was conducted on a cohort of normal kidney and ccRCC. ITH was assessed using DNA methylation (5mC) and CNV-based entropy and Euclidian distances. We found elevated 5mC heterogeneity and entropy in ccRCC relative to normal kidney. Variable CpGs are highly enriched in enhancer regions. Using intra-class correlation coefficient analysis, we identified CpGs that segregate tumor regions according to clinical phenotypes related to tumor aggressiveness. SETD2 wild-type tumors overall possess greater 5mC and copy number ITH than SETD2 mutant tumor regions, suggesting SETD2 loss contributes to a distinct epigenome. Finally, coupling our regional data with TCGA, we identified a 5mC signature that links regions within a primary tumor with metastatic potential.ConclusionTaken together, our results reveal marked levels of epigenetic ITH in ccRCC that are linked to clinically relevant tumor phenotypes and could translate into novel epigenetic biomarkers.

Oncohistones and disrupted development in pediatric-type diffuse high-grade glioma.

Recurrent, clonal somatic mutations in histone H3 are molecular hallmarks that distinguish the genetic mechanisms underlying pediatric and adult high-grade glioma (HGG), define biological subgroups of diffuse glioma, and highlight connections between cancer, development, and epigenetics. These oncogenic mutations in histones, now termed "oncohistones", were discovered through genome-wide sequencing of pediatric diffuse high-grade glioma. Up to 80% of diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG) and diffuse glioma arising in other midline structures including thalamus or spinal cord, contain histone H3 lysine 27 to methionine (K27M) mutations or, rarely, other alterations that result in a depletion of H3K27me3 similar to that induced by H3 K27M. This subgroup of glioma is now defined as diffuse midline glioma, H3K27-altered. In contrast, histone H3 Gly34Arg/Val (G34R/V) mutations are found in approximately 30% of diffuse glioma arising in the cerebral hemispheres of older adolescents and young adults, now classified as diffuse hemispheric glioma, H3G34-mutant. Here, we review how oncohistones modulate the epigenome and discuss the mutational landscape and invasive properties of histone mutant HGGs of childhood. The distinct mechanisms through which oncohistones and other mutations rewrite the epigenetic landscape provide novel insights into development and tumorigenesis and may present unique vulnerabilities for pHGGs. Lessons learned from these rare incurable brain tumors of childhood may have broader implications for cancer, as additional high- and low-frequency oncohistone mutations have been identified in other tumor types.

MinerVa: A high performance bioinformatic algorithm for the detection of minimal residual disease in solid tumors

How to improve the performance of circulating tumor DNA (ctDNA) signal acquisition and the accuracy to authenticate ultra low-frequency mutation are major challenges of minimal residual disease (MRD) detection in solid tumors. In this study, we developed a new MRD bioinformatics algorithm, namely multi-variant joint confidence analysis (MinerVa), and tested this algorithm both in contrived ctDNA standards and plasma DNA samples of patients with early non-small cell lung cancer (NSCLC). Our results showed that the specificity of multi-variant tracking of MinerVa algorithm ranged from 99.62% to 99.70%, and when tracking 30 variants, variant signals could be detected as low as 6.3 × 10 -5 variant abundance. Furthermore, in a cohort of 27 NSCLC patients, the specificity of ctDNA-MRD for recurrence monitoring was 100%, and the sensitivity was 78.6%. These findings indicate that the MinerVa algorithm can efficiently capture ctDNA signals in blood samples and exhibit high accuracy in MRD detection.

Clinical implications of myeloid malignancy‑related somatic mutations in aplastic anemia

Aplastic anemia (AA) is a potentially fatal bone marrow failure syndrome characterized by a paucity of hematopoietic stem cells and progenitor cells with varying degrees of cytopenia and fatty infiltration of the bone marrow space. Recent advances in genomics have uncovered a link between somatic mutations and myeloid cancer in AA patients. At present, the impact of these mutations on AA patients remains uncertain. We retrospectively investigated 279 AA patients and 174 patients with myelodysplastic syndromes (MDS) and performed targeted sequencing of 22 genes on their bone marrow cells using next-generation sequencing (NGS). Associations of somatic mutations with prognostic relevance and response to treatment were analyzed. Of 279 AA patients, 25 (9.0%) patients had somatic mutations, and 20 (7.2%) patients had one mutation. The most frequently mutated genes were ASXL1(3.2% of the patients), DNMT3A (1.8%) and TET2 (1.8%). In the MDS group, somatic mutations were detected in 120 of 174 (69.0%) patients, and 81 patients (46.6%) had more than one mutation. The most frequently mutated genes were U2AF1 (24.7% of the patients), ASXL1 (18.4%) and TP53 (13.2%). Compared with MDS patients, AA patients had a significantly lower frequency of somatic mutations and mostly one mutation. Similarly, the median variant allele frequency was lower in AA patients than in MDS patients (6.9% vs. 28.4%). The overall response of 3 and 6 months in the somatic mutation (SM) group was 37.5% and 66.7%, respectively. Moreover, there was no significant difference compared with the no somatic mutation (N-SM) group. During the 2-years follow-up period, four (20%) deaths occurred in the SM group and 40 (18.1%) in the N-SM group, with no significant difference in overall survival and event-free survival between the two groups. Our data indicated that myeloid tumor-associated somatic mutations in AA patients were detected in only a minority of patients by NGS. AA and MDS patients had different gene mutation patterns. The somatic mutations in patients with AA were characterized by lower mutation frequency, mostly one mutation, and lower median allelic burden of mutations than MDS. Somatic mutations were a common finding in the elderly, and the frequency of mutations increases with age. The platelet count affected the treatment response at 3 months, and ferritin level affected the outcome at 6 months, while somatic mutations were not associated with treatment response or long-term survival. However, our cohort of patients with the mutation was small; this result needs to be further confirmed with large patient sample.

Abstract LB077: Analysis of indel and structural variant error profiles in deep next generation sequencing data

Abstract Background: Accurate detection of low frequency mutations is of critical importance in the study of genetic heterogeneity, such as on the detection of minimal residual diseases for leukemias. Our prior work has resulted in successful error suppression for substitutions (10−4-10−5). However, the error profiles of indels and structural variants (SVs) remain elusive. Results: In this work, we generated ultra-deep sequencing data using our previously established dilution models (COLO829) on known somatic indels (n=23) and SVs (n=17). We discovered that the error rate of indels (10−6) and SVs (<2 × 10−7) are 100- to >1000-fold lower than that of SNVs. This finding was fully recapitulated in our analysis of 347 indels and 1248 SVs discovered from a relapsed B-ALL cohort of 103 patients, although homopolymer indels can have high error rates (>1%). We then performed a comprehensive study of homopolymer indels in 361 cancer driver genes by using whole genome data from 1662 healthy donors from the SJLIFE cohort. Our data indicated that the number of repeating units are highly predictive relative to the error rate of homopolymer indels (R2=0.988, p=4.89 × 10−8). Utilizing these insights, we assayed end-of-induction remission samples from 72 B-cell lymphoblastic leukemia patients that relapsed by selecting ~5 somatic clonal SNV/Indel/SV markers, which confirmed that SVs and indels have >10-fold lower error rates than SNVs. Our next generation sequencing (NGS) approach had 44 positive detections (61%) and outperformed the current standard method of clinical flow cytometry (n=37; 51%) for detecting minimal residual disease. The NGS-based method detected 92% of designed markers for samples with MRD>0.3%, and this detection rate dropped to 27% for MRD between 0.1% and 0.01%, indicating the difficulty in recovering mutant molecules when their frequencies are very low. Conclusions: Overall, we established indel and SV error profiles in deep next generation sequencing data enabling superior tumor detection performance at very low burdens, with lower error rates than what is observed for SNVs. Our work will have a significant impact on the clinical diagnosis and monitoring of human cancers and beyond. Citation Format: Ying Shao, Quang Tran, Pandurang Kolekar, Yanling Liu, Andrea McBride, Tyler Jones, Heather Mulder, Lingyun Ji, Benjamin Huang, Soheil Meshinchi, Jeffery Klco, Jinghui Zhang, William Carroll, Mignon Loh, Patrick Brown, John Easton, Xiaotu Ma. Analysis of indel and structural variant error profiles in deep next generation sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB077.

Abstract LB254: Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation

Abstract Sensitive and specific detection of low frequency mutations in acute myeloid leukemia (AML) is critical in research of minimal residual disease (MRD). At variant allele frequencies (VAF) below approximately 1%, PCR and sequencing errors result in prohibitive signal-to-noise ratios with next generation sequencing (NGS). Duplex Sequencing (DS) relates the original top and bottom DNA strands to make double stranded consensus sequences to greatly reduce errors. In two experiments we show further improvement of DS of AML-related genes by the use of enzymatic fragmentation (EF) and an updated gene panel that incorporates 2022 European LeukemiaNet (ELN) recommendations. In the first DS study, 29 AML-related genes (59 kb) were targeted in hybrid capture. Mutant cell line DNA was mixed into DNA from a healthy young donor to simulate MRD. Expected VAFs in mixtures were 1.0-0.003%. Samples comprised a mix of 4 insertions and deletions (indel), a mix of 15 single nucleotide variants (SNV), and 4 serial dilutions of a FLT3 ITD plus an NPM1 insertion. Pure diluent DNA was used as a negative control. Each sample was prepared in quadruplicate with mechanical fragmentation (MF) vs EF. DNA input mass was 1,500 ng per replicate, except for 50-250 ng for the FLT3/NPM1 mixes with expected VAF of 1% and 0.1%, respectively. Input masses were set to ensure >95% probability of detection of all mutations in the combined data. All targeted variants down to 0.003% VAF were detected in spike-in mixtures, with expected vs observed VAF highly correlated whether using MF or EF (R2 >0.98 for all mixtures). Duplex molecular depth was 1.2-2.0x higher with EF vs MF across input masses. Panel-wide mean duplex depth per 1,500 ng replicate was 30,376x for MF and 48,036x for EF, for combined mean depths of 121,508x for MF and 192,144x for EF. In the negative control, background mutational calls at spike-in positions were 4/2,993,429 duplex bases using MF and 0/4,780,491 duplex bases with EF, reflecting increased specificity with EF. Next, a revised panel targeting 36 AML-related genes (80 kb) was used for DS with EF. Here differing mutant cell line DNA was mixed into the same diluent from above (the negative control). This mixture harbors 27 variants with expected VAFs of 0.125-0.006% (4 indels, 21 SNVs, a FLT3 ITD and an NPM1 insertion), 17 of which (2 indels, 14 SNVs and the NPM1 insertion) span VAFs of 0.011-0.009% to establish a limit of detection of 0.01%. With 2,000 ng (+/- 10%) input, for 14 samples, panel-wide mean duplex depth was 56,528x. For variants with expected VAF ≥ 0.009%, the assay had >98% sensitivity, >96% specificity, and >98% accuracy at genomic positions of known true positive variants. In summary, DS with EF yields more data per nanogram of DNA than MF, maximizing the use of precious samples. This method exhibits extremely low background mutation signal and a low limit of detection across targets that are valuable in AML MRD research. Citation Format: Elizabeth Schmidt, Devon M. Fitzgerald, Camila Zanette, Gavin D. Meredith, Mark A. McElwain, Raul Burciaga, Kevin C. Vavra, Thomas H. Smith, Jesse J. Salk, Jake Higgins. Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB254.

Abstract LB078: pbfusion: Detecting gene-fusion and other transcriptional abnormalities using PacBio HiFi data

Abstract Sarcomas are a broad group of soft tissue and bone cancers that can be difficult to treat leading to a high mortality rate. Sarcomas comprise two broad genomic classes: (1) simple karyotypes, where a single oncogenic structural variant (SV) clonally expands a subtype that is diagnostic and relevant to tumor burden tracking; and (2) complex karyotypes, genomic instability, where SVs continuously arise throughout tumor evolution resulting in heterogeneous cellular subtypes. Class two sarcomas are harder to characterize using genome sequencing because there may be multiple low-frequency mutations. In both genomic classes, accurate and sensitive detection of fusion transcripts is needed to interpret functional consequences, to understand tumor biology and evolution, and potentially identify new targets for therapy. Many fusions have complex structures that cannot be uniquely resolved using short reads due to a lack of exon connectivity. PacBio full-length RNA isoform sequencing resolves complex fusions, providing more accurate breakpoints, and a complete sequence readout of the associated fusion transcript. To date, long-read fusion detection software was designed for high-error sequencing. PacBio HiFi data provides both full-length transcripts and accurate base calls. Here we present a fusion detection tool, pbfusion, specifically designed for HiFi sequence data, and apply it to sarcoma patients from both classes. pbfusion converts mapped sequences (either HiFi reads or Iso-Seq isoforms) into transcript objects that are annotated with reference gene models. Annotations determine whether transcripts are discordantly mapped, overlap differing genes, strand swap, transcriptional readthrough, or contain novel exons. The discordant exonic boundaries are treated as breakpoints between two genomic locations. All breakpoints are clustered with a multi-directional chaining algorithm and annotated with exonic information, gene names, and quality information. To test our method, we applied pbfusion to twelve samples from 8 sarcoma patients from both genomic classes. We discovered the known and novel fusions, including validated driver events in the fusion-driven samples (e.g. ASPSCR1-TFE3 in alveolar soft part sarcoma and SS18-SSX2/1 fusion in synovial sarcoma). This approach demonstrates the utility of HiFi sequence data for identification of fusion transcripts in patient samples, and the use of pbfusion in quantifying and annotating these events. pbfusion provides a user-friendly interface, can process a sample in a few minutes, and is freely available to the research community on Bioconda. Citation Format: Roger Volden, Zev Kronenberg, Aaron Gillmor, Ted Verhey, Michael Monument, Donna Senger, Harsharan Dhillon, Jason Underwood, Elizabeth Tseng, Daniel Baker, Primo Baybayan, Michael A. Eberle, Jonas Korlach, Sorana Morrissy. pbfusion: Detecting gene-fusion and other transcriptional abnormalities using PacBio HiFi data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB078.

Abstract LB004: Ultra-sensitive targeted DNA panel for very low-frequency mutation detection in circulating cell-free DNA

Abstract Due to its noninvasive nature and being readily available in peripheral blood and other bodily fluids, analysis of cell-free DNA (cfDNA) has become a promising application in cancer diagnosis, prognosis and treatment monitoring. However, disease-relevant cfDNA is present in a very limited amount in the huge background of normal cfDNA. This remains a challenge for any detection technology. Many currently available target enrichment and library preparation methods use regular DNA polymerase and amplification processes that introduce substantial bias and artifacts. This results in artifactual errors that greatly limit the detection of true low-frequency variants below 0.5% in heterogeneous samples, such as cfDNA. Here, we present a new targeted cfDNA workflow that overcomes challenges such as biases and artifacts. The workflow uses a highly optimized, high-fidelity reaction chemistry and incorporates UMIs into a single gene-specific, primer-based targeted enrichment process. Compared to regular DNA polymerase, this high-fidelity chemistry resulted in a five- to ten-fold decrease in base substitution error. An individual cancer panel was designed to specifically cover cancer-relevant hotspots and copy number genes with a dense primer design to accommodate the short length of cfDNA. Due to its high-fidelity chemistry and optimal panel design, our streamlined workflow can be completed in a single day. We also report consistent panel performance across different samples. Detection sensitivity and specificity were evaluated on a reference cfDNA sample and a simulated cfDNA sample by mixing enzyme-digested Genome in a Bottle samples, NA12878 and NA24385. We achieved close to 90% detection sensitivity and above 99.9% specificity for 0.1% variant. In addition, copy number variation could be successfully detected with a 1.5-fold difference over normal control samples. These results demonstrate an efficient targeted cfDNA panel workflow that enables cancer-relevant mutation detection with high sensitivity and accuracy. The applications presented here are for research use only. Not for use in diagnostic procedures. Citation Format: Michelle Baird, Mariam Ashraf, Emil Chistensen, Christa Haldrup, Zhong Wu, Eric Lader. Ultra-sensitive targeted DNA panel for very low-frequency mutation detection in circulating cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB004.

Low frequency of knockdown resistance mutation (L1014F) and the efficacy of PBO synergist in multiple insecticide-resistant populations of Anopheles gambiae in Ikorodu, Lagos State, Nigeria.

We evaluated the susceptibility status of Anopheles gambiae in two communities of Ikorodu, Lagos, Nigeria to DDT, deltamethrin, lambda cyhalothrin and bendiocarb. Anopheles immature stages were collected from their habitats in the surveyed community and allowed to emerge before exposure adult females to discriminating doses of WHO insecticides including DDT, deltamethrin, lambda cyhalothrin, bendiocarb and malathion. PBO synergistic bioassay was conducted for insecticides where the mosquito samples showed resistance. PCR assay was used for the detection of kdr mutation in the mosquitoes. Resistance to DDT (40% and 86%) and lambda cyhalothrin (75% and 84%) in Oke-Ota and Majidun respectively. Suspected resistance to deltamethrin (94.9%) and bendiocarb (93.5%) was recorded in Oke-Ota community and the mosquitoes were susceptible to malathion in both communities. KDR mutation (L1014F) from resistance samples from both locations though with a low frequency that significantly departs from Hardy-Weinberg's probability (P> 0.01). PBO synergized bioassay was able to increase knockdown, percentage mortality and restore full susceptibility to deltamethrin and bendiocarb. Results from this study indicates that the metabolic resistance mechanism is highly implicated in the resistance to different classes of insecticide in Ikorodu and this should be taken into consideration when implementing vector control activities in this area.

An institutional review of genomic sequencing in pediatric solid tumors.

Although tumor genomic profiling has aided the advancement of targeted genetic therapy, its clinical integration remains a challenge in pediatric cancers due to lower mutation frequency and less available targeted drugs. There have been multiple novel studies examining molecular sequencing in pediatrics; however, many of these studies primarily utilized large-scale, genome-wide screening applications that limit applicable use due to the availability of testing. This study examined the institutional use of a targeted, clinically available approach to tumor genomic sequencing. A retrospective chart review was performed on pediatric patients with solid tumors who were managed at Roswell Park Comprehensive Cancer Center and underwent molecular testing of their tumor biopsy via OmniSeq from August 2016 to July 2021. Results were reviewed for mutations considered to be "actionable" by targeted therapy. Patients with actionable mutations were further examined to evaluate treatment course, receival of targeted therapy, and clinical outcomes. We identified 64 pediatric patients consisting of 20 (31%) with CNS tumors and 44 (69%) with non-CNS tumors, ranging in age from 9 months to 21 years. Thirty-five total actionable mutations were identified amongst 27 patients (42%). Of these 27, 12 patients (44%) received at least 1 targeted drug against a respective actionable mutation, of which 6 patients (50%) achieved clinical benefit to therapy, including 1 complete response. The use of a clinically focused and readily available targeted molecular sequencing panel identified actionable mutations at a comparable rate to the large-scale, less readily available sequencing panels utilized in other studies. Half of our patients who received targeted therapy achieved a complete response or clinical benefit from therapy. Although targeted therapy has a role in pediatric cancer treatment, many newer drugs require further research on their safety and efficacy.

Abstract 6530: Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs

Abstract Background: The presence of circulating tumor DNA (ctDNA) can be used for minimal residual disease (MRD) monitoring and guide adjuvant therapy decisions. However, even when present, total ctDNA could be less than 0.01% of cell free DNA (cfDNA). Sequencing such low mutation frequency can be costly due to high sequencing read depth and accuracy requirements. The ability to enrich for low-abundance variants could improve detection performance and greatly lower sequencing cost by reducing the sequencing depth required. Purpose: The KAPA HyperPETE (Primer Extension Target Enrichment) workflow employs primer extension reactions to specifically capture and release target library molecules for sequencing, detects all major somatic variant types (SNV, InDel, CNV, MSI status, and novel fusion transcript partners) and could be leveraged to specifically target certain alleles. In this study, we assessed the variant enrichment capabilities of the KAPA HyperPETE workflow. Methods: Variant specific primers were designed to 8 low allele frequency single nucleotide variants (SNVs) in mixed cell line samples (NA12878 and NA24143, two Epstein-Barr virus transformed B cell female cell lines, with variant allele frequency (VAF) 1%, 0.5%, and 0.1%). Variant enrichment primers were used alongside a 37kb panel in the KAPA HyperPETE workflow. Additional optimizations and a study enriching all low allele frequency SNVs* in SeraCare ctDNA Mutation Mix v2 reference samples (VAF 1%, 0.5%, and 0.125%) are planned. Results: The preliminary results exhibited a median of 249✕, 159✕, and 86.5✕ enrichment of the targeted variants compared to the expected VAFs of 0.1%, 0.5%, and 1%, respectively. All 8 targeted variants were detected at 100K, 150K, and 550K read pairs per variant for VAF 1%, 0.5%, and 0.1%, respectively. Results from further optimizations and the study using SeraCare ctDNA samples will be shared at the AACR meeting. Conclusion: Preferential enrichment of specific variants down to 0.1% can be achieved using the KAPA HyperPETE Workflow. Utilizing primers designed for variant enrichment, we simultaneously enriched multiple variants and demonstrated the potential to minimize required sequencing. The KAPA HyperPETE workflow can be a powerful, cost-effective tool for detecting low frequency mutations in MRD research applications. KAPA HYPERPETE is For Research Use Only. Not for use in diagnostic procedures. KAPA HYPERPETE is a trademark of Roche. *Except GNA11 (COSM52969) due to stretches of polybase. Seraseq ctDNA Mutation Mix v2 gene list available on SeraCare website. Citation Format: Florence K. Crary-Dooley, Liu Xi, Ruben van der Merwe, Nitya M. Furtado, Jingchuan Li, Junyan Lin, Manuel Ochoa, Brian Godwin. Preferential low frequency allele enrichment with lower sequencing depth requirement using KAPA HyperPETE specialized primer designs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6530.

Abstract 4897: PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands

Abstract Adenoid Cystic Carcinoma (ACC) is a rare but aggressive malignancy of salivary gland, associated with protracted clinical course and fatal outcome. Treatment modalities are restricted to surgery and/or adjuvant radiotherapy and patients develop recurrence and distant metastasis over time. The absence of effective systematic therapy makes it incurable in advanced stage. ACC display an overall low mutation frequency, therefore very few actionable genetic alterations critical to the ACC development have been recognized. MYB fusion/overexpression is the most frequently found genetic alteration in ACC and is present in ~70% patients. This is followed by activating NOTCH mutations, reported in ~20% patients. Despite the presence of few targetable genetic alterations, recent studies revealed substantial transcriptomic heterogeneity amongst ACC tumors suggesting the role of epigenetic alterations in ACC oncogenesis. Symmetric dimethylation is an important epigenetic mechanism that regulates mRNA splicing, transcription, translation, cell cycle and oncogenic signaling pathways. Protein Arginine Methyl Transferase 5 (PRMT5) is a predominant enzyme for symmetric dimethylation among a family of 9 methyl transferases. In was suggested that PRMT5 plays a role in regulating tumor progression via modulation of MYC signaling, cancer stemness, and a wide array of additional cellular and transcriptional pro-oncogenic processes. While inhibition of PRMTs showed anti-oncogenic response in several preclinical tumor models and a subset of patients with advanced solid malignancies (including ACC), preclinical studies investigating the effect of PRMT5 blockade in ACC remain inadequate. In part, due to the scarcity of ACC specimens and limited availability of the experimental models. PRT543 is a selective and potent small molecule PRMT5 inhibitor that specifically targets PRMT5 among 37 methyl transferases. In the present study, we have investigated the effect this novel agent using a unique collection of ACC cell lines, organoids, and patient derived xenograft mouse models. To our knowledge, this is the first study investigating the therapeutic effect of PRT543 in several preclinical models of ACC. Specifically, we found that PRT543 has potent antitumor activity in in-vitro and in-vivo models with MYB expression and activating NOTCH mutations. Further, based on these observations, we have sequenced a collection of 50 ACC tumor samples to identify the subset of patients who may potentially benefit from PRT543 treatment based on their underlying genetic signatures. This study provides evidence underscoring the role of PRMT5 signaling in ACC and supports the clinical development of PRMT5 inhibitors for this indication. Citation Format: Vasudha Mishra, Alka Singh, Xiangying Chen, Michael Korzinkin, Claudia Wing, Viktoria Sarkisova, Alexandra Ozerova, Oksana Glushchenko, Venkat Thodima, Koichi Ito, Peggy Scherle, Mark Lingen, Rifat Hasina, Alexander Pearson, Ari Rosenberg, Alex Zhavoronkov, Bruce Ruggeri, Nishant Agrawal, Evgeny Izumchenko. PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4897.

Abstract 6608: High-sensitivity detection of specific ultra low-frequency somatic mutations for minimal residual disease monitoring

Abstract Minimal/Molecular residual disease (MRD) refers to the small number of tumor cells which may remain within a patient after therapeutic intervention. The detection of these remnants and monitoring of their abundance is a promising prognostic marker to identify individuals at risk of recurrence or in need of adjuvant therapy. Due to the low abundance of ctDNA present in samples obtained during remission, MRD assays need to be highly sensitive. In addition, each individual will have a different set of somatic variants, requiring personalized solutions for detection. Therefore, personalized NGS assays with high sensitivity and specificity are needed for MRD diagnostics. To address this need and empower accurate assessments of MRD, Twist Bioscience has developed the MRD Rapid 500 Panels. This product enables customers to design, manufacture and ship fully personalized MRD panels (up to 500 targets) in as little as six days. To demonstrate the detection sensitivity of Twist Rapid 500 MRD panels, we designed five custom MRD panels which specifically target somatic variants found in Breast, Lung, CRC, Melanoma and Renal Cell Carcinoma. Each of these MRD panels were designed to include 197 targets, with 3-5 variants per tissue origin and a selection of passenger mutations. To create the sample set, we blended synthetic variant sequences with fragmented cell line gDNA (NA12878) to form a contrived specimen which approximates the profile cell-free and circulating tumor DNA. Five frequency levels were created with average VAFs of 0% (WT), 0.01%, 0.05%, 0.1% and 2%. Libraries were prepared with UMI adapters and target enrichment was performed using the MRD panels. With a sequencing depth of 80,000x, variant calling results revealed that an average of 20 SNV targets can be detected with confidence in the 0.01% VAF samples for each MRD panel, clearly distinguishable from the WT control samples. In addition to demonstrating the accuracy of variant calling, we showcase the utility of targeting a large number of variants for the detection of an MRD signature at very low levels (e.g. 0.01% VAF). In summary, the performance of the Twist MRD Rapid 500 Panels showed high detection sensitivity of ultra low-frequency somatic mutations while maintaining high specificity for the purposes of MRD monitoring. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Tong Liu, Michael Bocek, Patrick Cherry, Shawn Gorda, Jean Challacombe, Derek Murphy, Esteban Toro. High-sensitivity detection of specific ultra low-frequency somatic mutations for minimal residual disease monitoring. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6608.

Abstract 6522: Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform

Abstract Ovarian and endometrial cancers come within the top-4 for incident cancers as well as deaths in North American women. Cure rates have not improved in 30 years as high-grade subtypes continue to be diagnosed in Stage III/IV. Attempts at early diagnosis have failed because high-grade cancer cells exfoliate and metastasize while the primary cancer is small and undetectable by existing tests based on imaging and blood-based tumour markers. DOvEEgene (Detecting Ovarian and Endometrial cancers Early using genomics) is a genomic uterine pap test developed by a McGill team to screen and detect these cancers while they are confined to the gynecologic organs and curable by surgery. The test identifies pathogenic somatic mutations in uterine brush samples A high sensitivity error-reducing capture technology (DOvEEgene-SureSelectHS) utilizing duplex sequencing interrogates the exons of 23 genes involved in the development of sporadic and hereditary ovarian and endometrial cancers. We apply a combination of germline gene panel testing on saliva samples with deep duplex sequencing to detect somatic mutations at <0.1% VAF, interrogation of microsatellite loci for instability and low coverage WGS for copy number analysis of uterine brush samples. Currently, DOvEEgene is the only test that can discriminate ovarian and endometrial cancers in peri- and postmenopausal women from benign gynecologic diseases common in that age group. This is important because pathogenic somatic driver mutations are also associated with increasing age and benign disease. DOvEEgene incorporates a deep machine-learning derived classifier that can discriminate the mutational signature of these cancers from benign disease aiming for a sensitivity of 70% and a specificity of 100% in a population with high background mutational burden. Here we tested the Onso system, a highly accurate sequencing technology from PacBio in order to potentially increase sensitivity while driving down sequencing costs by reducing required sequencing depth vs the current NGS standard. We sequenced 15 duplex Illumina sequencing libraries produced using the DovEE assay at PE 100bp mode and compared Onso data in non- duplex sequencing mode as well as duplex sequencing mode to the original duplex sequencing method. Here, we present this comparison and highlight the benefits of high accuracy sequencing for the detection of very low frequency (<0.1%) somatic mutations. Citation Format: Jiannis Ragoussis, Nairi Pezeshkian, Lucy Gilbert. Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6522.

Evaluation of the evolutionary genetics and population structure of Culex pipiens pallens in Shandong province, China based on knockdown resistance (kdr) mutations and the mtDNA-COI gene

BackgroundMosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China.MethodsAdult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene.ResultsTotally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations.ConclusionsThese data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts

Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low-throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. We developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a two-dimensional plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, the absolute quantification of different KRAS and GNAS variants was determined. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 fine-needle aspiration samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized by using template DNA at a concentration as low as 1 to 10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false-positive findings using high-fidelity polymerase, allowing for the detection of various KRAS and GNAS mutations with high probability in small amount of cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the fine-needle aspiration needle flush and microscopic lesions in resected specimens, were successfully identified. The proposed method allows for low-cost, accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.