Low Frequency Of Somatic Mutations Research Articles

From driver genes to gene families: A computational analysis of oncogenic mutations and ubiquitination anomalies in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a widespread primary liver cancer with a high fatality rate. Despite several genes with oncogenic effects in HCC have been identified, many remain undiscovered. In this study, we conducted a comprehensive computational analysis to explore the involvement of genes within the same families as known driver genes in HCC. Specifically, we expanded the concept beyond single-gene mutations to encompass gene families sharing homologous structures, integrating various omics data to comprehensively understand gene abnormalities in cancer. Our analysis identified 74 domains with an enriched mutation burden, 404 domain mutation hotspots, and 233 dysregulated driver genes. We observed that specific low-frequency somatic mutations may contribute to HCC occurrence, potentially overlooked by single-gene algorithms. Furthermore, we systematically analyzed how abnormalities in the ubiquitinated proteasome system (UPS) impact HCC, finding that abnormal genes in E3, E2, DUB families, and Degron genes often result in HCC by affecting the stability of oncogenic or tumor suppressor proteins. In conclusion, expanding the exploration of driver genes to include gene families with homologous structures emerges as a promising strategy for uncovering additional oncogenic alterations in HCC.

Low-frequency somatic mutations are heritable in tropical trees Dicorynia guianensis and Sextonia rubra

Somatic mutations potentially play a role in plant evolution, but common expectations pertaining to plant somatic mutations remain insufficiently tested. Unlike in most animals, the plant germline is assumed to be set aside late in development, leading to the expectation that plants accumulate somatic mutations along growth. Therefore, several predictions were made on the fate of somatic mutations: mutations have generally low frequency in plant tissues; mutations at high frequency have a higher chance of intergenerational transmission; branching topology of the tree dictates mutation distribution; and exposure to UV (ultraviolet) radiation increases mutagenesis. To provide insights into mutation accumulation and transmission in plants, we produced two high-quality reference genomes and a unique dataset of 60 high-coverage whole-genome sequences of two tropical tree species, Dicorynia guianensis (Fabaceae) and Sextonia rubra (Lauraceae). We identified 15,066 de novo somatic mutations in D. guianensis and 3,208 in S. rubra, surprisingly almost all found at low frequency. We demonstrate that 1) low-frequency mutations can be transmitted to the next generation; 2) mutation phylogenies deviate from the branching topology of the tree; and 3) mutation rates and mutation spectra are not demonstrably affected by differences in UV exposure. Altogether, our results suggest far more complex links between plant growth, aging, UV exposure, and mutation rates than commonly thought.

High Sensitivity Detection of Specific Ultra Low-Frequency Somatic Mutations for Minimal Residual Disease (MRD) Monitoring

High Sensitivity Detection of Specific Ultra Low-Frequency Somatic Mutations for Minimal Residual Disease (MRD) Monitoring

Clonal haematopoiesis of indeterminate potential in patients with venous thromboembolism.

Over the last decade, the concept of Clonal haematopoiesis of undetermined potential (CHIP) has emerged. Low frequency somatic mutations in hematopoietic cells can occur with age and might allow formation of clones in individuals with no characterized haematological pathology. These CHIP mutations are associated with an increased risk of cancer or atherothrombosis, and their prevalence are more and more studied in pathologies with an inflammatory component. In our study, we analysed, by next generation sequencing, the prevalence of CHIP mutation in 94 patients with deep venous thrombosis (DVT), distinguishing two clinical phenotypes: provoked distal and non-provoked proximal DVTs. We show that there is no difference in CHIP prevalence between these two groups, nor with a matched-aged control group. The number of mutation per patients and the affected genes remain also the same between the three groups. Consequently and despite the relative small number of patients in each cohort, it seems that CHIP is not a strong concern in venous thromboembolism.

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.

An anoikis-based signature for predicting prognosis in hepatocellular carcinoma with machine learning.

Background: Hepatocellular carcinoma (HCC) is a common malignancy with high mortality worldwide. Despite advancements in diagnosis and treatment in recent years, there is still an urgent unmet need to explore the underlying mechanisms and novel prognostic markers. Anoikis has received considerable attention because of its involvement in the progression of human malignancies. However, the potential mechanism of anoikis-related genes (ANRGs) involvement in HCC progression remains unclear. Methods: We use comprehensive bioinformatics analyses to determine the expression profile of ANRGs and their prognostic implications in HCC. Next, a risk score model was established by least absolute shrinkage and selection operator (Lasso) Cox regression analysis. Then, the prognostic value of the risk score in HCC and its correlation with clinical characteristics of HCC patients were further explored. Additionally, machine learning was utilized to identify the outstanding ANRGs to the risk score. Finally, the protein expression of DAP3 was examined on a tissue microarray (TMA), and the potential mechanisms of DAP3 in HCC was explored. Results: ANRGs were dysregulated in HCC, with a low frequency of somatic mutations and associated with prognosis of HCC patients. Then, nine ANRGs were selected to construct a risk score signature based on the LASSO model. The signature presented a strong ability of risk stratification and prediction for overall survival in HCC patients.Additionally, high risk scores were closely correlated with unfavorable clinical features such as advanced pathological stage, poor histological differentiation and vascular invasion. Moreover, The XGBoost algorithm verified that DAP3 was an important risk score contributor. Further immunohistochemistry determined the elevated expression of DAP3 in HCC tissues compared with nontumor tissues. Finally, functional analyses showed that DAP3 may promote HCC progression through multiple cancer-related pathways and suppress immune infiltration. Conclusion: In conclusion, the anoikis-based signature can be utilized as a novel prognostic biomarker for HCC, and DAP3 may play an important role in the development and progression of HCC.

Noninvasive detection of brain gliomas using plasma cell-free DNA 5-hydroxymethylcytosine sequencing.

Liquid biopsy techniques based on deep sequencing of plasma cell-free DNA (cfDNA) could detect the low-frequency somatic mutations and provide an accurate diagnosis for many cancers. However, for brain gliomas, reliable performance of these techniques currently requires obtaining cfDNA from patients' cerebral spinal fluid, which is cumbersome and risky. Here we report a liquid biopsy method based on sequencing of plasma cfDNA fragments carrying 5-hydroxymethylcytosine (5hmC) using selective chemical labeling (hMe-Seal). We first constructed a dataset including 180 glioma patients and 229 non-glioma controls. We found marked concordance between cfDNA hydroxymethylome and the aberrant transcriptome of the underlying gliomas. Functional analysis also revealed overrepresentation of the differentially hydroxymethylated genes (DhmGs) in oncogenic and neural pathways. After splitting our dataset into training and test cohort, we showed that a penalized logistic model constructed with training set DhmGs could distinguish glioma patients from healthy controls in both our test set (AUC=0.962) and an independent dataset (AUC=0.930) consisting of 111 gliomas and 111 controls. Additionally, the DhmGs between gliomas with mutant and wild-type isocitrate dehydrogenase (IDH) could be used to train a cfDNA predictor of the IDH mutation status of the underlying tumor (AUC=0.816), and patients with predicted IDH mutant gliomas had significantly better outcome (P=.01). These results indicate that our plasma cfDNA 5hmC sequencing method could obtain glioma-specific signals, which may be used to noninvasively detect these patients and predict the aggressiveness of their tumors.

Abstract LB063: Duplex Sequencing reveals ubiquitous clonal hematopoiesis and complex donor-recipient clonal dynamics following HSCT

Abstract Clonal hematopoiesis (CH aka CHIP) is the process whereby otherwise healthy individuals accumulate clones bearing low-frequency somatic mutations in hematologic malignancy (HM) driver genes. Historically thought to be a phenomenon of the elderly that is associated with increased risk of HM and cardiovascular disease, CH has been observed in increasingly younger cohorts as sequencing technology has advanced. CH in hematopoietic stem cell transplant (HSCT) donors has also been associated with adverse recipient outcomes. In this retrospective analysis, we asked whether CH mutations preferentially expanded in recipients vs. donors in paired samples collected a median of 34 years post-HSCT (range 6-45). 16 donor-recipient pairs were analyzed, 11 of which had a donor sample collected pre-HSCT. All individuals were healthy at the time of contemporary sampling. Median donor age was 26 years (range 12-84) at time of HSCT. We took advantage of the ultra-low error rate (<1-in-10-million) of Duplex Sequencing (DS) to comprehensively assess CH in genomic DNA from blood. We targeted 29 genes recurrently mutated in acute myeloid leukemia (AML) plus 48 kilobases of randomly chosen neutral genomic regions. The mean DS error-corrected molecular depth across all samples was 26,731x for AML-associated genes and 32,812x in the neutral regions. CH was detected in 100% of donors and recipients, with a trend toward more CH clones in older vs. younger donors. We defined CH as mutations observed in at least 2 unique DNA molecules, rather than by a threshold variant allele frequency (VAF). Among the 11 donors with matched historical and contemporary DNA samples, CH mutations in the latter had a higher average VAF and comprised more unique mutations than in the former, reflecting clonal expansions over time. The magnitude of CH expansions was greater for the AML-associated genes than for neutral loci, consistent with positive selection. Post-HSCT the overall number of unique mutations was similar between donors and recipients. Most donor CH variants did not preferentially expand in the recipient. A minority of recipients had a modestly increased overall average VAF relative to paired donors in AML genes. While the VAF of some likely pathogenic variants increased in the recipient vs the donor, this was not the case for most CH mutations in most recipients and the reciprocal was sometimes observed. DS represents a powerful tool for the high-resolution study of CH evolution. In a unique historical cohort comprising longitudinal samples from some of the earliest patients and donors to undergo HSCT, we observed limited instances of accelerated CH in recipients relative to donors. While this cohort is biased due to enriched inclusion of long-term survivors, our data exemplifies how healthy hematopoiesis, indistinguishable between donors and recipients, can be maintained almost half a century following transplant. Citation Format: Masumi Ueda, Jake Higgins, Thomas Smith, Elizabeth Schmidt, Fang Y. Lo, Charles C. Valentine, Jesse J. Salk, Rainer Storb, Jerald P. Radich. Duplex Sequencing reveals ubiquitous clonal hematopoiesis and complex donor-recipient clonal dynamics following HSCT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB063.

6. Emerging clinically-relevant applications of ultra-sensitive mutation detection

As the cost of next-generation sequencing decreases, ultra-deep targeted sequencing (UTS) provides an efficient approach for identifying low-frequency somatic mutations, allowing for earlier detection of the tumorigenic events responsible for cancer initiation and progression. We developed and experimentally validated a UTS-based pipeline to detect very-low-frequency mutations with in-silico error suppression. The potential utilities of ultra-sensitive mutation detection have been demonstrated in several recent clinically-relevant studies: 1) by comparing de novo mutations of individual-matched sun-exposed and non-sun-exposed normal skin areas, we identified a number of mutational features specifically associated with UV, including a 'mutation exempt' genomic region in TP53 that are almost never mutated during aging in non-sun-exposed skin but become highly mutable in sun-exposed skin [1]; 2) in a study of pelvic washing collected during radical-cystectomy of bladder cancers, we found the levels of residual tumor cells in pelvic washing are associated with tumor histology, and might be a potential biomarker for predicting cancer recurrence [2]; 3) to understand the mechanism of low-level tumor contamination in the histologically normal tissue adjacent to tumor, we designed and performed a special-collection procedure and found the tumor contamination in normal tissue was associated with inappropriate handling during pathology grossing and tissue procurement [3].

Assessing the Role of BCOR/BCORL1 and Other Historically Low-Frequency Somatic Mutations in Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of malignancies that are characterized by aberrant hematopoiesis and consequently abnormal blood counts. Patients with MDS have an increased likelihood of developing acute myeloid leukemia (AML). Next generation sequencing (NGS) is currently being used to study the genetic landscapes of diverse cancer types in finer detail than has previously been possible, owing to its ease-of-use and widespread availability. Crucially, the accuracy and broad breadth of genomic coverage provided by NGS approaches may permit the study of mutations that are rare in MDS patients, yet well studied in AML or other cancer types. Based on the fact that much less is known about particular rare somatic mutations in MDS, we decided to investigate 6 genes. IDH1 and IDH2 are targetable in patients with AML. Mutations in JAK2 are found in high proportions in patients with myeloproliferative neoplasms (MPNs) or MDS/MPN overlap neoplasms. Finally, other mutations in genes such as BCOR, BCORL1, and MUTYH also occur in MDS patients with appreciable-but generally low-frequencies such that their overall impact on MDS patients is largely unknown.Methods: We utilized the Tempus LENS platform to retrospectively characterize 236 MDS patients who underwent genomic-testing with the Tempus|xT assay (DNA-seq of 595-648 genes at 500x coverage; full transcriptome RNA-seq; data accessed on 06/28/2021). The patient population was 28% female, with a median age of 71.6 years. Mutations identified included germline and/or somatic single nucleotide variants (though we only considered somatic variants in this study), insertions/deletions and copy number variations (gains defined as ≥8 copies). For a subset of patients processed at Rush University Medical Center (RUMC), we additionally performed a curated clinical investigation into laboratory variables, prognostic data, treatment history and outcomes data.Results: Within the Tempus MDS dataset, we observed a range of mutation frequencies. In total, 58 patients had mutations in at least one of either: JAK2, BCOR, IDH2, IDH1, BCORL1, and MUTYH. When comparing the observed frequencies against values that have previously been reported, mutations in these 6 genes occurred with higher frequency in our dataset while most other mutations were in accordance with the relative frequencies reported by other sources (Table 1). We observed the largest deviation from expectation for BCOR. Whereas prior reports list this mutation as occurring with a frequency of <5%, greater than 10% of the records we analyzed contained a mutation in either BCOR (n=24, 10.16%) or BCORL1 (n=6, 2.54%). Four records in the dataset had mutations in both BCOR and BCORL1; a total of 26 records (11%) thus contained mutations in one of these two genes. Of these 26 patients, 50% were female and the median age was 70 years. The most commonly observed co-mutations with BCOR/BCORL1 were-in order of frequency-RUNX1 (50%), ASXL1 (23%), DNMT3A (23%), SF3B1 (19%), and STAG2 (19%). Additional data were derived from detailed clinical investigation into a limited subset of 4 patients who were treated at RUMC and who had mutations in either BCOR or BCORL1. Revised International Prognostic Scoring System (IPSS-R) scores were: high/very high (2) and low (2). Three of the four patients displayed complex karyotypes and three of the four progressed to AML.Conclusions: We identified a higher frequency of BCOR/BCORL1 mutations among patients with MDS than has been previously reported. At the same time, most other mutation frequencies were in relative accordance with existing reports. We observed complex cytogenetic abnormalities in 75% of patients for which we had extensive clinical information. Our data suggest an association between BCOR/BCORL1 mutations and complex karyotypes, a finding that may establish a prognostic role of BCOR/BCORL1 and have potential therapeutic implications. This hypothesis is, however, subject to further investigation and will require larger sample sizes that are likely to become available with the rapidly increasing utilization of next generation sequencing techniques and availability of high-quality, curated clinical information. [Display omitted] DisclosuresHockenberry: Tempus Labs, Inc: Current Employment, Current holder of stock options in a privately-held company. Okeke: Tempus Labs, Inc: Current Employment. Mahon: Tempus Labs, Inc: Current Employment. Hassane: Tempus Labs, Inc: Current Employment. Enschede: Tempus Labs, Inc: Current Employment, Current holder of stock options in a privately-held company. Shammo: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Astra zeneca: Research Funding; CTI pharma: Research Funding; Stemline therapeutics: Research Funding; Kartos Pharma: Research Funding; Takeda: Consultancy, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Honoraria, Research Funding; Abbvie: Current holder of individual stocks in a privately-held company, Research Funding; Baxter: Current holder of stock options in a privately-held company; sanofi: Consultancy, Honoraria, Speakers Bureau; NS Pharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Apellis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Consultancy, Honoraria, Research Funding, Speakers Bureau.

The Microenvironment of Small Intestinal Neuroendocrine Tumours Contains Lymphocytes Capable of Recognition and Activation after Expansion.

Simple SummaryThe body‘s immune system can recognize tumors because they often contain proteins that are either different from or more abundant than in normal cells. Here, we characterised the immune cells of a rare tumor type called small-intestinal neuroendocrine tumors (SINET). We find that so called tumour-infiltrating lymphocytes (TILs) can be grown in the laboratory and activated by challenging them with digested tumor. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.Traditionally, immune evasion and immunotherapy have been studied in cancers with a high mutational load such as melanoma or lung cancer. In contrast, small intestinal neuroendocrine tumours (SINETs) present a low frequency of somatic mutations and are described as genetically stable tumours, rendering immunotherapies largely unchartered waters for SINET patients. SINETs frequently metastasise to the regional lymph nodes and liver at the time of diagnosis, and no curative treatments are currently available for patients with disseminated disease. Here, we characterised the immune landscape of SINET and demonstrated that tumour-infiltrating lymphocytes (TILs) can be expanded and activated during autologous tumour challenge. The composition of lymphocyte subsets was determined by immunophenotyping of the SINET microenvironment in one hepatic and six lymph node metastases. TILs from these metastases were successfully grown out, enabling immunophenotyping and assessment of PD-1 expression. Expansion of the TILs and exposure to autologous tumour cells in vitro resulted in increased T lymphocyte degranulation. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.

A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

BackgroundNext-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency.ResultsAllele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%.ConclusionsFor properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

Systematic Investigation of mRNA N6-Methyladenosine Machinery in Primary Prostate Cancer

Background Appreciable findings have pointed out pivotal roles of N6-methyladenosine (m6A) machinery in cancer onset and progression. However, limited efforts have been directed towards relevant research in the prostate cancer area. Methods A PubMed search was conducted to acquire components of the mRNA m6A machinery. Multiomics integration was performed to systematically investigate the mRNA m6A machinery in primary prostate cancer. Furthermore, RNA interference assays of two prognostic m6A readers EIF3D and HNRNPA2B1 were conducted to explore m6A dependence of their functions in prostate cancer cell proliferation and migration. Results A total of 41 mRNA m6A regulators have been identified to date. A small degree of copy number aberrations and an extremely low frequency of somatic mutations were observed in the regulators across prostate tumors. Enrichment of CpG sites and extensive changes of DNA methylation in the m6A machinery were also found. Impact of copy number variation on m6A regulator expression was stronger than that of DNA methylation disturbance. Furthermore, our study identified a set of m6A regulators related to clinical features and/or survival which were largely m6A-binding proteins. The translation initiation factor subunit EIF3D and the splicing factor HNRNPA2B1 can be independent prognostic factors which may contribute to retardation and promotion of cancer progression, respectively, through affecting cancer-related processes such as cell cycle. Moreover, in vitro assays demonstrated that m6A impacted the EIF3D and HNRNPA2B1 roles in proliferation and migration of prostate cancer cells. Conclusions Our report systematically described molecular features of the mRNA m6A machinery and their potential roles in primary prostate cancer. Knowledge gained from this work may pave the way for further studies on the m6A system in prostate cancer.

Abstract 3114: Analytical validation of Illumina's TruSight Oncology 500 ctDNA assay

Abstract While tissue biopsies and imaging techniques remain the current standard of care, minimally invasive liquid biopsy tests are emerging as compelling complements in the clinical management of cancer patients. The ability to accurately detect low frequency somatic mutations, tumor mutational burden (TMB) and microsatellite instability (MSI) biomarkers from a single plasma sample using a broad-based NGS panel is of high value for biomarker discovery, detection and monitoring. Herein we report the results of our analytical validation of Illumina's TruSight Oncology 500 (TSO500) ctDNA assay which employs a hybrid-capture approach for target enrichment of 523 clinically-relevant cancer genes with unique molecular indices (UMIs) to enable detection of low frequency variants, CNVs, DNA fusions, TMB and MSI analysis. To characterize performance of small variant detection (SNVs and indels) and TMB analysis using the TSO500 ctDNA assay, we utilized various contrived reference standards with &amp;gt;1,500 unique variants below 5% variant allele frequency (VAF) and over 50 uncharacterized diseased and 20 healthy plasma samples. Cell-free DNA (cfDNA) isolates from healthy donors were used to evaluate the false positive (FP) rate as well as to evaluate potential impacts of blood collection tube (BCT) type on variant calling. For the tube type comparison, we procured 5 matched sets of healthy donor blood in both EDTA and Streck cfDNA BCTs. Where possible, unexpected somatic variants detected in healthy donor samples were orthogonally confirmed using droplet digital PCR (ddPCR). Additionally, to evaluate robustness of input on variant detection and TMB precision, cfDNA isolates from late stage cancer patients were tested at varying input amounts (10, 20, 30ng) in the TSO500 ctDNA assay. Analytical sensitivity, specificity and positive predictive value (PPV) were evaluated using reference standards. We observed &amp;gt;99% sensitivity for detecting SNVs and &amp;gt;98% for indels at or above 0.5% VAF. Additionally, we observed very high specificity and PPV (&amp;gt;99%), with a false positive rate of &amp;lt; 2 FP per 1.3 Mb down to 0.2% VAF. Further support for high specificity was observed in healthy donor samples where 25/26 apparent false positive somatic variants (0.2-0.7% VAF) were confirmed using ddPCR. Robustness of input studies revealed comparable coverage depth and uniformity can be achieved with 20 - 30 ng of cfDNA. Precision of variant detection and TMB will be reported for a subset of the diseased plasma sample cohort. In conclusion, the TSO500 ctDNA assay enables robust detection of low frequency variants and other clinically relevant biomarkers in EDTA and Streck derived plasma samples with at least 20ng of cfDNA input. Citation Format: Kirsten C. Verhein, Gunjan Hariani, Stephanie B. Hastings, Patrick Hurban. Analytical validation of Illumina's TruSight Oncology 500 ctDNA assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3114.

Dual Deep Sequencing Improves the Accuracy of Low-Frequency Somatic Mutation Detection in Cancer Gene Panel Testing.

Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.

Development and Analytical Validation of a DNA Dual-Strand Approach for the US Food and Drug Administration–Approved Next-Generation Sequencing–Based Praxis Extended RAS Panel for Metastatic Colorectal Cancer Samples

A next-generation sequencing method was developed that can distinguish single-stranded modifications from low-frequency somatic mutations present on both strands of DNA in formalin-fixed paraffin-embedded colorectal cancer samples. We applied this method for analytical validation of the Praxis Extended RAS Panel, a US Food and Drug Administration-approved companion diagnostic for panitumumab, on the Illumina MiSeqDx platform. With the use of the TruSeq amplicon workflow, both strands of DNA from the starting material were interrogated independently. Mutations were reported only if found on both strands; artifacts usually present on only one strand would not be reported. A total of 56 mutations were targeted within the KRAS and NRAS genes. A minimum read depth of 1800× per amplicon is required per sample but averaged >30,000× at maximum multiplexing levels. Analytical validation studies were performed to determine the simultaneous detection of mutations on both strands, reproducibility, assay detection level, precision of the assay across various factors, and the impact of interfering substances. In conclusion, this assay can clearly distinguish single-stranded artifacts from low-frequency mutations. Furthermore, the assay is accurate, precise, and reproducible, can achieve consistent detection of a mutation at 5% mutation frequency, exhibits minimal impact from tested interfering substances, and can simultaneously detect 56 mutations in a single run using 10 samples plus controls.

Characterization of BCR-ABL Specific CD4+ T Cells and Their Role in Controlling Ph+ Acute Lymphoblastic Leukemia

Background: Patients with BCR-ABL+ acute lymphoblastic leukemia (Ph+ ALL) can experience significant disease reduction after induction chemotherapy, but subsequently incur adverse long-term outcomes, due to the presence of minimal residual disease (MRD). This suggests a need for novel therapeutic strategies to eliminate MRD. Checkpoint blockade has been considered of little benefit in ALL due to the overall low frequency of somatic mutations. However, the protein product of the BCR-ABL translocation region forms a tumor associated antigen (TAA). Prior studies have demonstrated that patients with Ph+ ALL harbor CD8+ and CD4+ T-cells specific for peptides derived from this junctional region, presented in MHCI or MHCII contexts. This suggests that relapse in Ph+ ALL may be countered by immunotherapeutic strategies that expand sufficient numbers of BCR-ABL-specific effector T-cells. The current study uses a murine model of Ph+ ALL to characterize effector cell types and mechanisms responsible for leukemia control and eradication. We also investigated the impact of commonly used chemotherapy agents on effector cell function, to determine the viability of a combination immunotherapy and vaccination approach at eliminating MRD after induction. Methods: Bone marrow cells from p19ARF-null C57BL/6 mice were transduced with a BCR-ABL expressing retrovirus. Transformed cells were injected into immunocompetent B/6 mice, resulting in a uniformly fatal leukemia in 20-25 days. We used a peptide: MHC-II tetramer to label endogenous CD4+ T-cells specific for a BCR-ABL-derived peptide ("BAp") presented in an MHC II I-Ab context (hereafter referred to as "BAp"-specific T-cells). Results: Previously, we found that BAp-specific CD4+ T-cells were elicited during leukemia development but were limited by regulatory-T-cells (Tregs) that were cross-reactive with endogenous ABL protein. Nonetheless, a heterologous vaccination strategy using BAp peptide, combined with dual CTLA-4 and PD1 checkpoint blockade, was able to extend survival and eradicate leukemia in a subset of mice. Prolonged survival correlated with the robust expression of an ensemble of antigen-presentation molecules by host leukemia cells, as well as the presence of polyfunctional, cytotoxic CD4+ BAp-specific T-cells. To formally evaluate the relative contributions of CD4+ vs. CD8+ T-cells, we next established leukemia and allowed it to progress after the selective depletion of CD4+, CD8+, or both CD4+ and CD8+ T-cells. All mice were also treated with nilotinib and PD-L1 checkpoint blockade. Depletion of CD4+ T-cells led to markedly shortened survival. In contrast, depletion of CD8+ T-cells had no effect, and the majority of mice survived long-term. As this confirmed that CD4+ T-cells were critical for leukemia control, we next characterized the impact of common induction chemotherapy regimens on this subset. Combinations of cytotoxic chemotherapies mimicking either a standard intensity Hyper-CVAD-like regimen or a reduced-intensity regimen were administered to naïve mice, followed by vaccination with BAp. Mice treated with a Hyper-CVAD-like regimen generated fewer numbers of BAp-specific CD4+ T-cells in response to subsequent vaccination. Moreover, scRNAseq analysis of activated CD4+ T-cells demonstrated that standard intensity regimens induced dysfunctional gene expression signatures. These changes were not observed after treatment with a reduced intensity regimen. Dysfunctional signatures were characterized by the upregulation of senescence-associated genes including p21, as well as decreased expression of T-stem-central-memory (Tscm) genes including TCF7 and Tox2. Altogether these findings suggest that Hyper-CVAD-like chemotherapy inhibits proliferation as well as the self-renewal of CD4+ T-cells responding to leukemia antigens. Conclusions: CD4+ T-cells specific for peptides derived from the BCR-ABL protein are responsive to PD-L1 checkpoint blockade, and are critical for Ph+ ALL clearance. Standard intensity induction chemotherapy leads to significant dysfunction of these cells. Ongoing experiments are exploring the mechanisms underlying this dysfunction, and methods to overcome it via BCR-ABL-based peptide vaccination combined with checkpoint blockade. Validation experiments utilizing human samples are also underway and will be reported. Disclosures No relevant conflicts of interest to declare.

Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Bi-allelic Loss of ENG or ACVRL1

Hereditary hemorrhagic telangiectasia (HHT) is a Mendelian disease characterized by vascular malformations (VMs) including visceral arteriovenous malformations and mucosal telangiectasia. HHT is caused by loss-of-function (LoF) mutations in one of three genes, ENG, ACVRL1, or SMAD4, and is inherited as an autosomal-dominant condition. Intriguingly, the constitutional mutation causing HHT is present throughout the body, yet the multiple VMs in individuals with HHT occur focally, rather than manifesting as a systemic vascular defect. This disconnect between genotype and phenotype suggests that a local event is necessary for the development of VMs. We investigated the hypothesis that local somatic mutations seed the formation HHT-related telangiectasia in a genetic two-hit mechanism. We identified low-frequency somatic mutations in 9/19 telangiectasia through the use of next-generation sequencing. We established phase for seven of nine samples, which confirms that the germline and somatic mutations in all seven samples exist in trans configuration; this is consistent with a genetic two-hit mechanism. These combined data suggest that bi-allelic loss of ENG or ACVRL1 may be a required event in the development of telangiectasia, and that rather than haploinsufficiency, VMs in HHT are caused by a Knudsonian two-hit mechanism.

Validation of CRC and NSCLC somatic mutations detected by NGS using ddPCR

Next Generation Sequencing (NGS) has become powerful tool in molecularoncology. It allows multiparallel targeted sequencing that enablescomprehensive assessment of tumor heterogeneity. Detection of mutations incolorectal cancer (CRC) and non-small cell lung cancer (NSCLC) definespatients diagnosis, therapy and prognosis. Multiple genes, their somaticmutations to be precise, carry different degrees of importance for each of theseaspects. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which wasused in this study, allows detection of hotspot mutations in 22 genes in a singlereaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequencymutation detection and it has been used as a validation tool for mutations thatwere detected with NGS. It has high sensitivity and enables accurate detection ofa mutant allele against a background of abundant wild type alleles. For this study35 samples of CRC and NSCLC were sequenced and selected samples wereanalysed with ddPCR for KRAS, NRAS, EGFR and BRAF genes. All theprocessed samples were successfully sequenced and had average base coverage&gt;500X. NGS sequencing proved itself to be cost effective, has shorterturnaround time and is highly sensitive. Out of 35 samples, 25 had geneticalterations, while 10 samples were reported as wild type but were still tested withddPCR as controls. In three samples low frequency somatic mutations weredetected with NGS and mutation frequencies were verified using ddPCR.

The Pathogenesis of Port Wine Stain and Sturge Weber Syndrome: Complex Interactions between Genetic Alterations and Aberrant MAPK and PI3K Activation.

Port wine stain (PWS) is a congenital vascular malformation involving human skin. Approximately 15–20% of children a facial PWS involving the ophthalmic (V1) trigeminal dermatome are at risk for Sturge Weber syndrome (SWS), a neurocutaneous disorder with vascular malformations in the cerebral cortex on the same side of the facial PWS lesions. Recently, evidence has surfaced that advanced our understanding of the pathogenesis of PWS/SWS, including discoveries of somatic genetic mutations (GNAQ, PI3K), MAPK and PI3K aberrant activations, and molecular phenotypes of PWS endothelial cells. In this review, we summarize current knowledge on the etiology and pathology of PWS/SWS based on evidence that the activation of MAPK and/or PI3K contributes to the malformations, as well as potential futuristic treatment approaches targeting these aberrantly dysregulated signaling pathways. Current data support that: (1) PWS is a multifactorial malformation involving the entire physiological structure of human skin; (2) PWS should be pathoanatomically re-defined as “a malformation resulting from differentiation-impaired endothelial cells with a progressive dilatation of immature venule-like vasculatures”; (3) dysregulation of vascular MAPK and/or PI3K signaling during human embryonic development plays a part in the pathogenesis and progression of PWS/SWS; and (4) sporadic low frequency somatic mutations, such as GNAQ, PI3K, work as team players but not as a lone wolf, contributing to the development of vascular phenotypes. We also address many crucial questions yet to be answered in the future research investigations.