Low Allele Frequency Research Articles

MICon Contamination Detection Workflow for Next-Generation Sequencing Laboratories Using Microhaplotype Loci and Supervised Learning

Innovation in sequencing instrumentation is increasing the per-batch data volumes and decreasing the per-base costs. Multiplexed chemistry protocols after the addition of index tags have further contributed to efficient and cost-effective sequencer utilization. With these pooled processing strategies, however, comes an increased risk of sample contamination. Sample contamination poses a risk of missing critical variants in a patient sample or wrongly reporting variants derived from the contaminant, which are particularly relevant issues in oncology specimen testing in which low variant allele frequencies have clinical relevance. Small custom-targeted next-generation sequencing (NGS) panels yield limited variants and pose challenges in delineating true somatic variants versus contamination calls. A number of popular contamination identification tools have the ability to perform well in whole-genome/exome sequencing data; however, in smaller gene panels, there are fewer variant candidates for the tools to perform accurately. To prevent clinical reporting of potentially contaminated samples in small next-generation sequencing panels, we have developed MICon (Microhaplotype Contamination detection), a novel contamination detection model that uses microhaplotype site variant allele frequencies. In a heterogeneous hold-out test cohort of 210 samples, the model displayed state-of-the-art performance with an area under the receiver-operating characteristiccurve of 0.995.

Reference population database for 24 STR loci of selected provinces of Argentina.

Argentinean population is the result of admixture between South Amerindians, Europeans and to a lesser degree, Africans. Since the advent of forensic molecular genetics, the construction of local reference databases became mandatory. Aiming to further extend the technical quality reference database of Argentina, we present herein the allele frequencies for 24 autosomal STRs, including D22S1045, and SE33 (not previously reported for Argentina in STRidER). Genotypes of 6454 unrelated individuals (3761 males and 2694 females) from 13 out of 23 provinces were analysed. Forensic parameters were calculated for each marker. The observed heterozygosity ranged from 0.661 (TPOX) to 0.941 (SE33). The locus SE33 was revealed to be the most informative marker showing the highest values for PIC (0.955), GD (0.952), TPI (8.455) and PE (0.879). On the other hand, TPOX turned out to be the least informative marker: PIC (0.618), GD (0.669), and PE (0.371). The high number of analyzed individuals allowed detecting low frequency alleles and microvariants in CSF1PO; D16S539 and D21S11 D18S51; PENTA D; PENTA E and at locus D6S1043. This study is the most extensive for Argentina and complements the already reported information concerning the autosomal STRs commonly used in forensic identification. The results were submitted passing STRidER quality control standards (QC), receiving the reference number STR000327 v.2.

Severe Acute Respiratory Syndrome Coronavirus 2 Delta Variant Genomic Variation Associated With Breakthrough Infection in Northern California: A Retrospective Cohort Study.

The association between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic variation and breakthrough infection is not well defined among persons with Delta variant SARS-CoV-2 infection. In a retrospective cohort, we assessed whether individual nonlineage defining mutations and overall genomic variation (including low-frequency alleles) were associated with breakthrough infection, defined as SARS-CoV-2 infection after coronavirus disease 2019 primary vaccine series. We identified all nonsynonymous single-nucleotide polymorphisms, insertions, and deletions in SARS-CoV-2 genomes with ≥5% allelic frequency and population frequency of ≥5% and ≤95%. Using Poisson regression, we assessed the association with breakthrough infection for each individual mutation and a viral genomic risk score. Thirty-six mutations met our inclusion criteria. Among 12 744 persons infected with Delta variant SARS-CoV-2, 5949 (47%) were vaccinated and 6795 (53%) were unvaccinated. Viruses with a viral genomic risk score in the highest quintile were 9% more likely to be associated with breakthrough infection than viruses in the lowest quintile, but including the risk score improved overall predictive model performance (measured by C statistic) by only +0.0006. Genomic variation within SARS-CoV-2 Delta variant was weakly associated with breakthrough infection, but several potential nonlineage defining mutations were identified that might contribute to immune evasion by SARS-CoV-2.

Suspected Germline TP53 Variants and Clonal Hematopoiesis of Indeterminate Potential: Lessons Learned From a Molecular Tumor Board.

Li-Fraumeni syndrome (LFS) is a pan-cancer predisposition syndrome caused by germline pathogenic variants in the gene TP53. The interpretation of TP53 variants in clinical scenarios outside the classic LFS criteria may be challenging. Here, we report a patient affected by 2 primary cancers at later ages, who harbored a likely pathogenic TP53 at low allele frequency detected in a blood sample. The Molecular Tumor Board committee at our institution revisited the case of a patient who was enrolled in a research protocol for the investigation of genetic conditions associated with neuroendocrine tumors. Clinical, familial, and molecular data were reviewed. The patient received germline testing using a next generation sequencing multi-gene panel and was incidentally found to harbor a TP53 likely pathogenic variant, with 22% of variant allele fraction. Additional samples, including a second blood sample, oral swab, and saliva, were collected for DNA analysis. A new TP53 sequencing round was performed with the attempt to distinguish between a true constitutional germline variant and a somatically acquired variant due to aberrant clonal expansion of bone marrow precursors. Patient's personal and familial history of cancer did not meet classic nor Chompret LFS criteria. Environmental risk factors for cancer were identified, such as alcohol abuse and tobacco exposure. The TP53 variant initially found in the next-generation sequencing was confirmed by Sanger sequencing in the previous DNA sample extracted from blood for the first analysis and in a second blood sample collected 6 years later. The TP53 variant was not detected in the DNA extracted from the oral swab and saliva samples. Considering the low TP53 variant allele fraction in blood, absence of variant detection in oral swab and saliva samples, the lack of LFS clinical criteria, and history of exposure to environmental risk factors for cancer, the main hypothesis for this case was aberrant clonal expansion due to clonal hematopoiesis. Oncologists should interpret TP53 findings during germline testing with caution.

The Mighty NUMT: Mitochondrial DNA Flexing Its Code in the Nuclear Genome.

Nuclear-mitochondrial DNA segments (NUMTs) are mitochondrial DNA (mtDNA) fragments that have been inserted into the nuclear genome. Some NUMTs are common within the human population but most NUMTs are rare and specific to individuals. NUMTs range in size from 24 base pairs to encompassing nearly the entire mtDNA and are found throughout the nuclear genome. Emerging evidence suggests that the formation of NUMTs is an ongoing process in humans. NUMTs contaminate sequencing results of the mtDNA by introducing false positive variants, particularly heteroplasmic variants present at a low variant allele frequency (VAF). In our review, we discuss the prevalence of NUMTs in the human population, the potential mechanisms of de novo NUMT insertion via DNA repair mechanisms, and provide an overview of the existing approaches for minimizing NUMT contamination. Apart from filtering known NUMTs, both wet lab-based and computational methods can be used to minimize the contamination of NUMTs in analyses of human mtDNA. Current approaches include: (1) isolating mitochondria to enrich for mtDNA; (2) applying basic local alignment to identify NUMTs for subsequent filtering; (3) bioinformatic pipelines for NUMT detection; (4) k-mer-based NUMT detection; and (5) filtering candidate false positive variants by mtDNA copy number, VAF, or sequence quality score. Multiple approaches must be applied in order to effectively identify NUMTs in samples. Although next-generation sequencing is revolutionizing our understanding of heteroplasmic mtDNA, it also raises new challenges with the high prevalence and individual-specific NUMTs that need to be handled with care in studies of mitochondrial genetics.

Detecting Low-Variant Allele Frequency Mosaic Pathogenic Variants of NF1, TSC2, and AKT3 Genes from Blood in Patients with Neurodevelopmental Disorders

Growing evidence indicates that early and late postzygotic mosaicism can cause neurodevelopmental disorders (NDDs), but detection of low variant allele frequency (VAF) mosaic variants from blood remains a challenge. Data of 2162 patients with NDDs who underwent conventional genetic tests were reviewed and a deep sequencing was performed using a specifically designed mosaic next-generation sequencing (NGS) panel in the patients with negative genetic test results. Forty-four patents with neurocutaneous syndrome, malformation of cortical development, or nonlesional epileptic encephalopathies were included. In total, mosaic variants were detected from blood in 1.2% (25/2162) of the patients. Using conventional NGS panels, 22 mosaic variants (VAF, 8.8% to 29.8%) were identified in 18 different genes. Using a specifically designed mosaicism NGS panel, three mosaic variants of the NF1, TSC2, and AKT3 genes were identified (VAF, 2.0% to 11.2%). Mosaic variants were found frequently in the patients who had neurocutaneous syndrome (2/7, 28.6%), whereas only one or no mosaic variant was detected for patients who had malformations of cortical development (1/20, 5%) or nonlesional epileptic encephalopathies (0%, 0/17). In summary, mosaic variants that contribute to the spectrum of NDDs can be detected from blood via conventional NGS and specifically designed mosaicism NGS panels, and detection of mosaic variants using blood will increase diagnostic yield.

Insecticide susceptibility and detection of kdr-gene mutations in Aedes aegypti of Peshawar, Pakistan

Dengue is an endemic disease in Peshawar, Pakistan and its primary vector is Aedes aegypti mosquito. Due to absence of vaccines and proper treatment for dengue, vector control becomes a necessary tool for disease management. Reported insecticide resistance in vectors is a serious threat to the control of dengue. This study presents the susceptibility status of Ae. aegypti to eight insecticides in district Peshawar along with one of the first attempts to screen mutations in the vector's knock down resistant gene (kdr). Local Ae. aegypti was found to be highly resistant to DDT and Deltamethrin while they were susceptible to Cyfluthrin and Bendiocarb. DNA sequencing of domains II and III of kdr-gene detected four SNPs in domain IIS6 at positions S989P and V1016G while two mutations were reported at position T1520I and F1534C in domain IIIS6. Lowest allele frequency was observed for S989P and V1016G positions while it was highest for F1534C. Among mutational combinations SSVVTICC (43%) was evidently the most predominant combination, where T1520I was heterozygous and F1534C was homozygous mutant. The study concludes instecticide resistance in local dengue population of Peshawar, Pakistan. The resistance observed is to some extent also corroborated in the molecular study of kdr gene. Findings herein can be utilized in designing dengue vector control strategies in Peshawar.

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 2289: Predicting tumor somatic versus clonal hematopoiesis origin for short variants in liquid assay

Abstract Introduction A common challenge presented by emerging liquid biopsy technology is to distinguish somatic variants originated in CH from those in tumor. While sequencing of genomic DNA from matched peripheral blood mononuclear cells (PBMC) can effectively identify CH variants, its wide application still remains prohibitively expensive. Computational-only solutions are challenging due to shared bioinformatic presentations in both CH and somatic variants such as low allele frequency. Development of such algorithm with clinically meaningful accuracy will have extensive utility in cancer care. Method We sequenced paired plasma-buffy coat samples from 754 pan-cancer patients using the FoundationOne® Liquid CDx (F1LCDx) assay. The samples were randomized into a training and a test set. Ground truth (tumor somatic vs. CH) for SV, including base substitutions and short indels, was determined by comparing variant calls in plasma vs in PBMC. A deep learning prediction model was developed in the training set. The model incorporates genomic and clinical features as input and returns a CH prediction score, which was dichotomized by a learned threshold. After the threshold and model parameters were finalized, we applied the model to the test set to evaluate performance. Results In the test set of 222 paired samples (523 CH and 904 tumor somatic variants), the model achieved 91% sensitivity (probability of predicting true CH variants as CH) and 88% specificity (probability of predicting true tumor somatic variants as tumor somatic). The model accurately identified CH variants in challenging genes such as TP53 (sensitivity = 94%, specificity = 90%, n = 144) and ATM (sensitivity = 93%, specificity = 95%, n = 49), which that are known to harbor both tumor somatic and CH variants. Performance was further evaluated by cancer types and variant allele frequency. CH variants were identified with 90% sensitivity and 89% specificity in non-small cell lung carcinoma (n = 384), and with 87% sensitivity and 100% specificity in pancreas cancer (n = 53). CH variants were identified with 92% sensitivity and 87% specificity in 1%-5% variant allele frequency (n = 645), and with 94% sensitivity and 94% specificity in 5%-10% variant allele frequency (n = 189). Of note, a well-known oncogenic mutation in ATM was predicted as CH in a prostate cancer patient, which would prevent ineffective administration of PARP inhibitors. Multiple plasma specimens also gained more accurate bTMB and tumor fraction estimation by filtering out CH variants. Conclusion For F1LCDx, we developed a computational method that distinguishes tumor somatic from CH variants from plasma cfDNA sequencing alone. The classifier exhibited high sensitivity and specificity in a pan-cancer cohort. Additional analytical validations are required in an external dataset. Clinical utility of this classifier will likely be further demonstrated in CH-corrected bTMB and tumor fraction. Citation Format: Daokun Sun, Zheng Kuang, Alexander D. Fine, Eliana Polisecki, Holley Gettler, Hawra Al-Rekabi, Dean Pavlick, Yanmei Huang, Lee A. Albacker, Brennan Decker, Chang Xu. Predicting tumor somatic versus clonal hematopoiesis origin for short variants in liquid assay [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 2289.

Abstract 6612: Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia

Abstract The cancer hallmark concept defines biological properties that play a key role in cancer development and progression. The hallmarks reflect generic properties across all cancer types, and are not directly quantifiable nor applicable in cancer diagnostics. Here, we applied a data-driven approach to identify quantifiable hallmarks to be used as a basis for precision cancer medicine (PCM) in Acute Myeloid Leukemia (AML). We applied deep exome, transcriptome, DNA methylation and proteome profiling as well as ex-vivo functional testing of 525 drugs to 118 AML patient samples. Unsupervised multi-omic dimensionality reduction defined 11 independent axes of biological variability that we interpreted to reflect data-driven hallmarks (DDHM) of AML. Each DDHM integrates and ranks different data types and features, pinpointing those molecular features that were most informative for each hallmark. We calculated values for the 11 DDHM for each patient, constructing a DDHM-based precision medicine approach for AML diagnosis and therapy assignment. Most DDHMs were driven by other data types than genomics. For several DDHMs, different cytogenetic and mutational drivers converge on the same hallmarks and specific drug vulnerabilities. We also see how DDHM predictors of poor prognosis and high-risk AML are distinct from those that dictate specific drug response vulnerabilities. DDHM 2 reflected the cell differentiation path leading towards resistance to BH3 mimetics, ACK-inhibitors, and anthracyclines, and sensitivity to MEK inhibitors and TLR8 agonists. DDHM 2 derived a strong impact from protein expression of GATA2, MLLT11, DNMT3B and from RNA expression of WT1 and SOX4 as well as from multiple markers of different monocytic subtypes. DDHM 5 depicted cell cycle regulation and the Megakaryocytic-erythrocyte progenitor cell state that was enriched in AML patients with antecedent hematological disease. Interestingly, DDHM5 was linked to the sensitivity to purine analogs and vinca alkaloids. Moreover, DDHMs 1 and 8 captured clinical risk groups, prognosis, and responsiveness to hypomethylating agents. These DDHMs also revealed the phenotypic and functional biology that distinguished patients with high versus low FLT3-ITD mutant allele frequency. Validation of the DDHMs is realized through profiling and analysis of prospective AML samples, AML cell lines and previously published datasets. In summary, we present a data-driven approach for defining hallmarks in AML. The application of the DDHMs in AML provides a new paradigm for PCM and an opportunity for combinatorial therapeutic targeting. Each patient is characterized by a combination of independent and potentially druggable hallmarks, as opposed to traditional stratification in PCM, where each patient is assigned to one specific subgroup defined by genetic or other biomarkers. Citation Format: Tom Erkers, Nona Struyf, Tojo James, Francesco Marabita, Mattias Vesterlund, Nghia Vu, Cornelia Arnroth, Albin Österroos, Anna Bohlin, Sofia Bengtzén, Matthias Stahl, Rozbeh Jafari, Lukas Orre, Yudi Pawitan, Brinton Seashore-Ludlow, Janne Lehtiö, Sören Lehmann, Päivi Östling, Olli Kallioniemi. Data-driven hallmarks of cancer as a new paradigm for precision medicine: multi-omics and functional profiling in acute myeloid leukemia. [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 6612.

Abstract 2297: Sensitive mutation detection by simultaneous targeted sequencing of the Watson and Crick strands

Abstract Background Detecting mutations in circulating cell-free DNA (ccfDNA) allows for non-invasive monitoring of tumors, however, is hampered by low fractions of tumor DNA and low efficiencies of current technologies. Methods We describe a new and simple method for simultaneous multiplex targeted next-generation sequencing (NGS) of the Watson and Crick strands. We also develop a custom bin-based algorithm and unique molecule identifier (UMI)-aware data algorithm to segregate and suppress background noises. The performance of the method was evaluated with a reference standard DNA that contains 44 mutations, and experimented with dilutions for low mutant allele frequencies (MAFs) and with various amounts of DNA inputs. We validated our methods using blood samples collected from lung cancer patients with various stages and at follow-ups. A commercial droplet digital PCR (ddPCR) for a seven-plex KRAS mutation detection assay was used as an orthogonal method to validate the sensitivity and specificity of our NGS methods. Results Using merely 1 ng of DNA (300 genome equivalent) containing 44 known mutations at 1% minor allele frequencies (MAFs) across 19 genes, our method detected 43 known and 0 false mutations, demonstrating a superior enrichment efficiency (> 99%) approaching the theoretical limit that misses target molecules owning to random sampling alone and high specificity. In the analysis of cfDNA obtained from plasma (1 mL per patient) of 77 lung cancer cases, mutations were identified in 47 (61%) of the cancer patients. Using the equal-volume (1 mL) aliquot plasma available from 70 patients (7 cases had insufficient samples), the ddPCR assay showed 6 samples were positive and 64 samples were negative. Among the 6 ddPCR-positive samples, all were tested positive by our NGS method, demonstrating similar sensitivity as the ddPCR assay while having the advantage of the capacity to test a panel of genes. Among the 64 ddPCR-negative samples, our method showed 63 were KRAS-negative and 1 was positive, indicating a specificity of 98.4%. With a detailed examination of the “false-positive” sample when using the ddPCR as the gold standard, the image of ddPCR result in fact showed two dots in the ‘grey zone’, indicating a possibility of a false-negative ddPCR result for the sample, rather than a false-positive in our NGS method. Using 1 mL of plasma, our method can detect mutation at 0.05% MAF and predict lung cancer relapse prior to current clinical methods. Conclusion Our new NGS method enables sensitive detection of mutations when only a trace amount of material is available and may be useful for cancer minimal residual disease monitoring. Citation Format: Firaol T. Kebede, Weiwei Bian, William C. Cho, Zongli Zheng. Sensitive mutation detection by simultaneous targeted sequencing of the Watson and Crick strands [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 2297.

Abstract 211: The performance characteristic of the low input tagmentation-based whole genome sequencing in high quality somatic variant calling

Abstract Whole genome sequencing (WGS) is widely used for cancer diagnostic and therapeutic applications in clinical practice and clinical trials. Currently, the Truseq PCR-free library preparation methods are routinely used in genomics laboratories. However, microgram amounts of DNA input is a limitation due to limited materials often from clinically-derived specimens. Here, we evaluated a novel low input (100-300 ng) PCR-free tagmentation (TAG) based library preparation for WGS. Replicates of TAG- and ligation-based (Illumina TruSeq) sequencing libraries were prepared from 3 pairs of breast cancer-derived tumors (HCC1195, HCC1143 and HCC1187) and matched B-lymphocyte-derived normal (HCC1195BL, HCC1143BL and HCC1187BL) cell lines. Libraries were sequenced on Illumina Novaseq 6000 platforms to target 30x and 90x mean coverage for normal and tumor samples respectively. Raw sequencing data were aligned to the hg38 by Isaac Aligner before variant analysis by Strelka2. Technical sequencing metrics demonstrated high similarity between TAG- and ligation-based workflows, including passing filter reads, Q30%, aligned reads, mean coverage. Germline variants of HCC1395BL showed greater than 98% precision and sensitivity using ligation-based variants as a reference. For somatic mutation calling from TAG-based libraries, HCC1395, HCC1143 and HCC1187, 88%, 85% and 86% precision and 81%, 70%, 68% sensitivity was observed, respectively, compared to the ligation-based method. Furthermore, using the SEQC2 Consortium high-confidence mutation set, TAG- and ligation- based variant calls of HCC1395 had 90% and 93% precision and 74% and 73% sensitivity, respectively with this high confidence reference. Further investigation showed that a high fraction of false negative calls was associated with low variant allele frequency (<10%). Compared to an available reference dataset for benchmarking somatic calling pipelines from New York Genome Center, TAG- and ligation-based variant calls of HCC1143 yielded 85% and 83% precision and 63% and 63% sensitivity, respectively; TAG- and ligation-based variant calls of HCC1187 resulted in 91% and 92% precision and 81% and 79% sensitivity, respectively. In summary, the low input TAG-based WGS protocol produced highly reproducible variant calls with highly concordant somatic variant calls compared to the commonly-used PCR-free ligation-based methods and to reference callset. Further evaluation is warranted to broaden clinical application from minimal starting material.The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Liqun Jiang, Xijun Zhang, Camille Alba, Gauthaman Sukumar, Elizabeth A. Rice, Matthew D. Wilkerson, Clifton L. Dalgard. The performance characteristic of the low input tagmentation-based whole genome sequencing in high quality somatic variant calling [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 211.

Abstract 5714: Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer

Abstract BACKGROUND: Cell-free circulating tumor DNA (ctDNA) assays have been adopted to monitor therapeutic response in both early- and late-stage cancer. However, tests currently available require deep-targeted sequencing to detect cancer-specific mutations at low mutant allele frequency (MAF) levels in the circulation. Recently, we developed a tumor-agnostic approach called DELFI Tumor Fraction (DELFI-TF), a Bayesian probabilistic model designed to predict plasma tumor fractions based on genome-wide fragmentation-related features. METHODS: Overall, 692 longitudinal plasma samples collected from 153 patients with initially unresectable colorectal cancer (CRC) liver-only metastases participating in the phase III study CAIRO5 (NCT02162563) were split across a training (RAS/BRAF mutant) and a validation cohort (RAS/BRAF wildtype). For all cell-free DNA (cfDNA) samples of patients within the training cohort (n=312), the tumor burden was quantified as the MAF of the tumor-tissue-proven RAS/BRAF variant measured by digital droplet PCR (ddPCR). Using cfDNA fragment-sequencing statistics from low-coverage WGS data, a Bayesian regression model was trained against the MAF of the tumor-specific driver RAS/BRAF variant in all longitudinal timepoints in the training cohort. Changes in DELFI-TF scores during first-line FOLFOX/FOLFIRI and bevacizumab therapy were assessed to predict disease response. A tissue-informed approach was applied to detect minimal residual disease in the subgroup of patients treated with curative intent (complete resection of the primary tumor and liver metastases). RESULTS: In the training cohort, DELFI-TF scores strongly correlated with RAS/BRAF MAF measured by ddPCR (r=0.85, p<0.001). DELFI-TF captured plasma tumor fractions associated with copy number changes when ddPCR failed to detect RAS/BRAF mutations. Before treatment initiation, DELFI-TF correlated with the sum of the largest diameters on CT scans according to RECIST1.1 (r=0.49, p<0.001). Patients with negative and positive changes in DELFI-TF more often exhibited response and progressive disease based on consecutive RECIST assessments, respectively. Median progression-free survival was 9.9 and 12.8 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.007). Overall survival was 28.8 and 58.9 months for patients with DELFI-TF low versus DELFI-TF high, respectively (p=0.001). In patients treated with curative intent, tissue-informed focal and arm-level copy number changes were detected 4-12 weeks after metastasectomy. Most patients presenting molecular relapse were later diagnosed with clinical recurrence by conventional CT imaging. CONCLUSIONS: DELFI-TF demonstrates the ability to use cfDNA fragmentomes to estimate cfDNA tumor burden with a performance comparable to standard approaches for treatment response monitoring and clinical outcome prediction. Citation Format: Bahar Alipanahi, Iris van 't Erve, Keith Lumbard, Laurel K. Millberg, Zach Skidmore, Lorenzo Rinaldi, Jacob Carey, Jennifer Tom, Cornelis J. A. Punt, Nicholas C. Dracopoli, Gerrit A. Meijer, Robert B. Scharpf, Victor E. Velculescu, Remond Fijneman, Alessandro Leal. Cell-free DNA fragmentation profiling for monitoring therapeutic response in metastatic colorectal cancer. [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 5714.

Abstract 1507: Multiregional sampling of high grade glioma identifies regional biologic signatures

Abstract High grade gliomas (HGG) are aggressive primary brain malignancies typified by diffuse invasion, genetic heterogeneity, and a universally fatal outcome. MRI-defined contrast-enhancing (CE) tumor burden serves as the clinical standard that guides maximal surgical resection and post-therapy response assessment. However, HGGs also comprise an invasive non-enhancing (NE) tumor margin that extends beyond the CE core and harbors the cells that contribute to recurrence. Sampling restrictions have hindered the comprehensive study of these NE HGG cell populations driving tumor progression. Herein, we present an integrated multi-omic analysis of 313 spatially matched multi-regional CE and NE tumor biopsies from 68 HGG patients, performing whole exome and RNA sequencing of both IDH wild-type and IDH mutant HGGs. We report spatially restricted molecular profiles in IDH-mutant HGG, highlighting a concern for sampling bias given the importance of molecular diagnosis and prognostication in IDH-mutant HGG. Regardless of IDH status, we found that NE tumor regions harbored the highest proportion of private mutations, which reflects an increased development of regional genomic complexity in infiltrative tumor. The multiregional genomic profiling of our IDH wild-type HGG cohort reveals that EGFR and NF1 somatic alterations occur as mutually exclusive events in 98.7% of tumors. However, we resolved rare low allele frequency co-alterations of EGFR and NF1 within the NE region. We find this co-occurrence enriched in recurrent tumors, pointing to the early emergence of NF1 inactivation in the NE regions. We constructed genomic models predictive of recurrent disease from both NE and CE regions, which highlight the occurrence of clonal EGFR copy number alterations and NF1 loss as clonal or subclonal events, respectively, emphasizing the regional and temporal complexity of well-studied canonical driver alterations. We detailed the spatially unique acquisition of multiple distinct EGFR alterations giving rise to intra-tumoral EGFR mosaicism, a challenge in the implementation of EGFR directed therapies. Our study also identified two transcriptomic clusters delineated by the significant overrepresentation of neuronal (NEU) and glycolytic/plurimetabolic (GPM) pathway-based functional states in the NE region. NE regions of the NEU subtype harbor the greatest proportion of private mutations, suggesting these infiltrative tumor cells accumulate alterations without clonal expansion. GPM populations conversely displayed a less branched phylogeny and were transcriptionally enriched in immune cell signatures. This phenotypic dichotomy between GPM and NEU populations supports the growing body of evidence that invasive GBM cells either take on a neuronal phenotype for active invasion or a more metabolic phenotype involving interaction with astrocytes, other glial cells, and infiltrating immune cells. Citation Format: Mylan R. Blomquist, Leland S. Hu, Fulvio D'Angelo, Taylor M. Weiskittel, Francesca P. Caruso, Shannon P. Fortin Ensign, Christopher Sereduk, Gustavo De Leon, Lee Curtin, Javier Urcuyo, Ashlynn Gonzalez, Ashley Nespodzany, Teresa Noviello, Jennifer M. Eschbacher, Kris A. Smith, Peter Nakaji, Bernard R. Bendok, Richard S. Zimmerman, Chandan Krishna, Devi Patra, Naresh Patel, Mark Lyons, Kliment Donev, Maciej Mrugala, Alyx Porter, Anna Lasorella, Kristin R. Swanson, Michele Ceccarelli, Antonio Iavarone, Nhan L. Tran. Multiregional sampling of high grade glioma identifies regional biologic signatures [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 1507.

Abstract 4343: Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to EGFR inhibitors

Abstract Two well-characterized EGFR inhibitor (EGFRi) therapies (cetuximab, panitumumab) have been FDA approved for metastatic colorectal cancer (CRC). Unfortunately, the extended RAS/RAF testing required in the drug labels, identifies only non-responders, and only ~50% of treated patients will respond to therapy. Thus, there is an unmet need to develop additional biomarkers to identify EGFRi sensitive patients. Using an innovative hybrid approach fusing gene expression and DNA sequencing, we recently reported that combined mutations in APC and TP53 were strongly correlated with a validated gene expression signature measuring cetuximab sensitivity in CRC human tumors (Yang et al. Cancer Epidemiol Biomarkers Prev. 2019), suggesting a potential positive biomarker role of APC + TP53 mutations. This finding prompted us to develop a DNA test designed to positively select EGFRi-responsive patients as augmentation to currently used negative predictors for CRC patients. By leveraging the TwinStrand Duplex Sequencing (DS) technology, we recently developed an ultrasensitive custom 6-gene panel (APC, TP53, KRAS, BRAF, NRAS, and HRAS) CLIA-certified assay for FFPE CRC tissues. The assay has been analytically validated using reference cell lines (n=10) which were individually sequenced to >3,000x Duplex depth. The 6-gene DS assay yielded exceptionally high assay performance: (1) accuracy (passed, r2 = 0.98 for Duplex VAF vs. target VAF for Positive Control mutations); (2) sensitivity (passed, all mutations >1/5,000 were detected in all Positive Control replicates; 1 mutation at 1/5,000 was detected in 2/3 replicates), (3) specificity (passed, 2 mutations detected in the Negative Control at 1 count in 1 replicate; VAF <1/10,000), and (4) precision (passed, <2-fold variation among Positive Control replicates for VAF >1/500). Importantly, the validation analysis on fresh frozen (FF):FFPE paired tissues from 21 CRC patients shows: (1) The 6-gene DS assay performed on FFPE samples faithfully reproduces assay results on FF samples; (2) The ultrasensitive DS assay can accurately detect additional “new” mutations at low allelic frequencies compared to a standard NGS method. TwinStrand DS identified 17 mutations not identified by standard NGS; 13 of these new mutations had < 10% VAF. Furthermore, preliminary Kaplan-Meier analysis of the third-line EGFRi data shows that while the statistical significance was not achieved due to the small simple size (n=28), patients harboring combined APC and TP53 mutations(AP) (vs non-AP patients) tended to have longer progression free survival (6.65 vs 3.95 months, P=0.064) and overall survival (17.80 vs 10.65 months, P=0.14). Additional data acquisition and analysis is ongoing. Thus, there is an opportunity to change clinical practice and standards of care to ultimately improve CRC outcomes if this new, robust, sensitive test is clinically-validated. Citation Format: Mingli Yang, Michael J. Schell, Lance Pflieger, Jesse Salk, Timothy J. Yeatman. Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to EGFR inhibitors. [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 4343.

5586004 INCREASE IN HEMOGLOBINOPATHIES CARRIAGE AMONG PREGNANT WOMEN IN GERMANY PREDICTED BY MIGRATION AND BIRTH DATA ANALYSIS

Background: Germany is one of the most densly populated countries in Europe, yet it has low allele frequencies for hemoglobinopathies in its population. According to estimates by Kohne et al. (2010), approx. 0.48%1 of Germany’s population are carriers for at least one hemoglobinopathy. Modell et al. (2007) estimated the proportion of carriers among pregnant women in Germany at 0.54%2. Given the sharp increase in migration between 2014 and 2016, predominantly from high prevalence countries for hemoglobinopathies (Figure 1), we expect a rise in carrier numbers among present German population. Although new-born screening for sickle cell disease has been introduces as standard of care in Germany in 2021, low awareness for hemoglobinopathies among general medical staff in general and the lack of existing routine screening in later life, e.g. during pregnancy, still pose a major challenge to monitor the development of these disorders and to ensure appropriate clinical care and consulting services.Aims: To estimate hemoglobinopathies carriage rates among pregnant women in Germany during the last decade. Methods: We extracted data on number and origin of foreign women at childbearing age (15 - 50 years) between 2010 and 2020 in Germany by federal state and year3 from the GENESIS database of the German federal statistical office. Using the published data by Modell and Darlison on carriage rates in pregnancies by country4, we calculated the proportion of women at childbearing age with risk for carrying a hemoglobinopathy per state and year. With the help of data on life births by mothers’ citizenship status (German or foreign)3, we calculated a fertility adjustment factor (= fertility rate of all foreign women/ total fertility rate in Germany, both per state and year) and estimated the yearly proportion of pregnancies at risk for carrying a hemoglobinopathy per state. Results: The estimated hemoglobinopathies carriage rates among pregnancies showed an increasing trend in all 16 German federal states in the last decade (Figure 2). The states Bremen, Hamburg, Berlin, and Hesse showed the highest carriage rates in 2020 with 3.84%, 2.78%, 2.66% and 2.64% respectively (HbS heterozygosity included). Notably, the states with lowest estimated carriage rates in 2020 (Thuringia, Saxony, Saxony-Anhalt and Brandenburg) showed the strongest increases compared to its initial rates in 2010 (Figures 2-4). On a national average, we calculated an increase of approximately 60% in pregnancies at risk for hemoglobinopathy carriage (Figure 5).Conclusion: Our analysis indicates a clear and strong increase in hemoglobinopathy carriage rates among pregnancies in all federal states in parallel to the increase in migration into Germany between 2014 and 2016. Further studies are required to verify these findings and to evaluate the necessity of new public health measures as prenatal screening and consulting services for hemoglobinopathies in Germany.

SvCapture: efficient and specific detection of very low frequency structural variant junctions by error-minimized capture sequencing.

Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to comparable strategies for rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from samples with known SV properties, we demonstrate that duplex sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of newly created SVs, suggesting end joining as a possible formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.

Genome-wide association study of a semicontinuous trait: illustration of the impact of the modeling strategy through the study of Neutrophil Extracellular Traps levels.

Over the last years, there has been a considerable expansion of genome-wide association studies (GWAS) for discovering biological pathways underlying pathological conditions or disease biomarkers. These GWAS are often limited to binary or quantitative traits analyzed through linear or logistic models, respectively. In some situations, the distribution of the outcome may require more complex modeling, such as when the outcome exhibits a semicontinuous distribution characterized by an excess of zero values followed by a non-negative and right-skewed distribution. We here investigate three different modeling for semicontinuous data: Tobit, Negative Binomial and Compound Poisson-Gamma. Using both simulated data and a real GWAS on Neutrophil Extracellular Traps (NETs), an emerging biomarker in immuno-thrombosis, we demonstrate that Compound Poisson-Gamma was the most robust model with respect to low allele frequencies and outliers. This model further identified the MIR155HG locus as significantly (P=1.4×10-8) associated with NETs plasma levels in a sample of 657 participants, a locus recently highlighted to be involved in NETs formation in mice. This work highlights the importance of the modeling strategy for GWAS of a semicontinuous outcome and suggests Compound Poisson-Gamma as an elegant but neglected alternative to Negative Binomial for modeling semicontinuous outcome in the context of genomic investigations.

Genome-wide association study of population-standardised cognitive performance phenotypes in a rural South African community

Cognitive function is an indicator for global physical and mental health, and cognitive impairment has been associated with poorer life outcomes and earlier mortality. A standard cognition test, adapted to a rural-dwelling African community, and the Oxford Cognition Screen-Plus were used to capture cognitive performance as five continuous traits (total cognition score, verbal episodic memory, executive function, language, and visuospatial ability) for 2,246 adults in this population of South Africans. A novel common variant, rs73485231, reached genome-wide significance for association with episodic memory using data for ~14 million markers imputed from the H3Africa genotyping array data. Window-based replication of previously implicated variants and regions of interest support the discovery of African-specific associated variants despite the small population size and low allele frequency. This African genome-wide association study identifies suggestive associations with general cognition and domain-specific cognitive pathways and lays the groundwork for further genomic studies on cognition in Africa.

Complexities of recapitulating polygenic effects in natural populations: replication of genetic effects on wing shape in artificially selected and wild-caught populations of Drosophila melanogaster.

Identifying the genetic architecture of complex traits is important to many geneticists, including those interested in human disease, plant and animal breeding, and evolutionary genetics. Advances in sequencing technology and statistical methods for genome-wide association studies have allowed for the identification of more variants with smaller effect sizes, however, many of these identified polymorphisms fail to be replicated in subsequent studies. In addition to sampling variation, this failure to replicate reflects the complexities introduced by factors including environmental variation, genetic background, and differences in allele frequencies among populations. Using Drosophila melanogaster wing shape, we ask if we can replicate allelic effects of polymorphisms first identified in a genome-wide association studies in three genes: dachsous, extra-macrochaete, and neuralized, using artificial selection in the lab, and bulk segregant mapping in natural populations. We demonstrate that multivariate wing shape changes associated with these genes are aligned with major axes of phenotypic and genetic variation in natural populations. Following seven generations of artificial selection along the dachsous shape change vector, we observe genetic differentiation of variants in dachsous and genomic regions containing other genes in the hippo signaling pathway. This suggests a shared direction of effects within a developmental network. We also performed artificial selection with the extra-macrochaete shape change vector, which is not a part of the hippo signaling network, but showed a largely shared direction of effects. The response to selection along the emc vector was similar to that of dachsous, suggesting that the available genetic diversity of a population, summarized by the genetic (co)variance matrix (G), influenced alleles captured by selection. Despite the success with artificial selection, bulk segregant analysis using natural populations did not detect these same variants, likely due to the contribution of environmental variation and low minor allele frequencies, coupled with small effect sizes of the contributing variants.