Low Allele Fraction Research Articles

Somatic mutations in autoinflammatory and autoimmune disease.

Somatic mutations (also known as acquired mutations) are emerging as common, age-related processes that occur in all cells throughout the body. Somatic mutations are canonically linked to malignant processes but over the past decade have been increasingly causally connected to benign diseases including rheumatic conditions. Here we outline the contribution of somatic mutations to complex and monogenic immunological diseases with a detailed review of unique aspects associated with such causes. Somatic mutations can cause early- or late-onset rheumatic monogenic diseases but also contribute to the pathogenesis of complex inflammatory and immune-mediated diseases, affect disease progression and define new clinical subtypes. Although even variants with a low variant allele fraction can be pathogenic, clonal dynamics could lead to changes over time in the proportion of mutant cells, with possible phenotypic consequences for the individual. Thus, somatic mutagenesis and clonal expansion have relevant implications in genetic testing and counselling. On the basis of both increased recognition of somatic diseases in clinical practice and improved technical and bioinformatic processes, we hypothesize that there will be an ever-expanding list of somatic mutations in various genes leading to inflammatory conditions, particularly in late-onset disease.

A novel liquid biopsy assay with an 88% detection rate of genomic alterations across CNS tumors.

2077 Background: Diagnosis and treatment decisions for CNS tumors rely upon imaging with subsequent histological and genomic analyses of tissue to identify appropriate courses of therapy. Genomic profiling of tumor tissue for informative alterations is used to guide treatment, however contemporaneous tissue can sometimes be unavailable, inconclusive or overlook possible actionable alterations given the high amount of spatial heterogeneity in certain tumor types such as glioblastomas GBMs. Liquid biopsies may pose a possible solution, as they are readily accessible and provide a holistic snapshot of the tumoral genomic landscape. However, detection of ctDNAs from CNS tumors is impeded by its inherently low shedding biology and poor permeability across the BBB resulting in low plasma variant allele fractions (VAFs), with alteration detection rates ranging 27-55%. Thus, current assays do not yet have the sensitivity to be clinically useful in aiding decision making for CNS tumors. Methods: 62 cases of primary CNS tumors were submitted commercially for assessment with Northstar Select, a tumor-naive, plasma-based ctDNA liquid biopsy assay. Cases were obtained from a single clinic dating from January-December 2023. Results: See table. Conclusions: There remains an unmet clinical need for a highly sensitive liquid biopsy assay for use in determining the genomic landscape of CNS tumors to aid in clinical decision making. Current assays do not have a low enough limit of detection (LoD) to accurately detect ctDNAs at low VAFs. Herein, we demonstrate that Northstar Select has a detection rate of 88.7% in all cases and 91.4% in GBMs, nearly doubling current rates. This is due to the assay’s uniquely low LoD of 0.13-0.16% VAF, which is below the 0.17% median VAF demonstrated across CNS tumors in this study, allowing for more low-abundance alterations to be detected. Given the increased total variant detection rate, nearly 50% of which were actionable when alterations were detected, these data support the clinical utility of Northstar Select to inform clinical decisions either as a complement to imaging and tissue analyses or independently when tissue biopsies are infeasible. These data further suggest that the clinical utility of Northstar Select may be expanded into metastatic brain disease and other low ctDNA shedding tumors. [Table: see text]

Somatic GNA11/GNAQ variants in a cohort of Chinese children with phakomatosis pigmentovascularis.

Postzygotic mutations in the GNAQ/GNA11 genes, which encode the G-protein nucleotide binding protein alpha subunits, have been identified in patients with phakomatosis pigmentovascularis (PPV). However, little is known about the Chinese population. To identify pathogenic mutations in pediatric patients with PPV within the Chinese population. We performed whole-exome sequencing (WES) using skin lesion tissues from pediatric patients diagnosed with PPV. Additionally, ultradeep-targeted sequencing was conducted to validate the somatic mutations. A genotype-phenotype correlation was analyzed by integrating data from previous reports with the findings of the present study. Thirteen patients were enrolled, all diagnosed with the cesioflammea type of PPV, except for one patient with an unclassifiable type. We identified somatic GNA11 c.547C>T (p.R183C) variant in seven patients and GNAQ c.548G>A (p.R183Q) in four patients, with low allelic fractions ranging from 2.1% to 8.6% through ultradeep sequencing. Besides, a GNAQ c.548G>A (p.R183Q) variant was detected through targeted sequencing in one of two patients who did not exhibit detectable variants via WES. The genotype-phenotype correlation analysis, involving 15 patients with a GNA11 variant and 10 with a GNAQ variant, revealed that facial capillary malformation (87% vs. 50%, P = 0.075) and ocular melanocytosis (80% vs. 40%, P = 0.087) appeared to be more frequent in patients with GNA11 mutation compared to those with GNAQ mutations. All four patients diagnosed with cesiomarmorata type or overlapping cesioflammea and cesiomarmorata type PPV carried the GNA11 variant. Our study demonstrated that the majority of PPV patients in the Chinese population carried a postzygotic variant of GNAQ/GNA11, thus further confirming the pathogenic role of GNAQ/GNA11 mosaicism in the development of PPV cesioflammea type.

The application of targeted RNA sequencing for the analysis of fusion genes, gene mutations, IKZF1 intragenic deletion, and CRLF2 overexpression in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is characterized by highly genetic heterogeneity, owing to recurrent fusion genes, gene mutations, intragenic deletion, and gene overexpression, which poses significant challenges in clinical detection. RNA sequencing (RNA-seq) is a powerful tool for detecting multiple genetic abnormalities, especially cryptic gene rearrangements, in a single test. Sixty samples (B-ALL, n = 49; T-ALL, n = 9; mixed phenotype acute leukemia (MPAL), n = 2) and 20 controls were analyzed by targeted RNA-seq panel of 507 genes developed by our lab. Of these, 16 patients were simultaneously analyzed for gene mutations at the DNA level using a next-generation sequencing panel of 51 genes. Fusion genes, CRLF2 expression, and IKZF1 intragenic deletion were also detected by reverse transcription-polymerase chain reaction (RT-PCR). Karyotype analysis was performed using the R-banding and G-banding technique on bone marrow cells after 24 hours of culture. Partial fusion genes were analyzed using fluorescence in situ hybridization (FISH). Compared with the results of Karyotype analysis, FISH, and RT-PCR, the detection rate of fusion genes by targeted RNA-seq increased from 48.3% to 58.3%, and six unexpected fusion genes were discovered, along with one rare isoform of IKZF1 intragenic deletion (IK10). The DNA sequencing analysis of 16 ALL patients revealed that 96.2% (25/26) of gene mutations identified at the DNA level were also detectable at the RNA level, except for one mutation with a low variant allele fraction. The detection of CRLF2 overexpression exhibited complete concordance between RT-PCR and RNA-seq. The utilization of RNA-seq enables the identification of clinically significant genetic abnormalities that may go undetected through conventional detection methods. Its robust analytical performance might bring great application value for clinical diagnosis, prognosis, and therapy in ALL.

Abstract 3492: Comprehensive analysis of hematological malignancies with optical genome mapping and Bionano VIA™Software

Abstract The current standard for analysis of samples with suspected hematological cancer is karyotyping, fluorescence in situ hybridization (FISH), and/or chromosomal microarray (CMA). However, these methods have several limitations for the detection of structural variants (SVs) or copy number variants (CNV) that are smaller than 5 Mbp (karyotyping). Additionally, they are limited in their ability to detect low allele fraction (LAF) events and cannot detect the absence of heterozygosity (AOH) or the loss of heterozygosity (AOH/LOH - FISH and karyotyping). Optical genome mapping (OGM) from Bionano can enable accurate detection of SV and CNV events of sizes down to 5 kbp, with high sensitivity to detect LAF events down to 5% variant allele fraction. Bionano’s VIA™ software provides visualization and analysis support for OGM SV events including an improved Circos plot and ideogram for whole genome review. VIA incorporates CNV calling from OGM data using the SNP-FASST3 algorithm which also allows for accurate copy number calling at allele fractions as low as 10%. CNV detection performance was evaluated using 280 CNV events ranging in size from 175 kbp to 8 Mbp on the 22 autosomal chromosomes. Sensitivity and PPV were calculated for each size range separately. Using SNP-FASST3, VIA also adds the ability to detect AOH/LOH events from OGM data. By analyzing locations where known SNPs disrupt the specific nucleotide sequence motif used by the DLE-1 labeling enzyme, a B-allele frequency can be calculated and enable AOH/LOH detection at low allelic fractions. AOH/LOH performance using SNP-FASST3 was evaluated using a cohort of constitutional and cancer samples analyzed with orthogonal methods as well as with simulated events. Sensitivity for 20-25 Mbp AOH events was 92% with PPV greater than 90% at 25% aberrant cell fraction. The VIA 7.0 release also includes the ability to create a customizable decision tree using supplied reference annotation files for CNV, AOH and SV event pre-classification. VIA resources include a panel of target variants derived from various medical societies for clinical analysis guidelines specific to Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) and an aggregation of all hematological malignancy guidelines (pan-heme). These methods, combined with tools for expert review and reclassification of variants, allow users to conduct whole genome interpretation and report results. These new features in VIA provide a streamlined workflow for analyzing heterogeneous hematological cancer samples and demonstrate the analytical performance of updated approaches for detection of CNV and AOH/LOH regions at low allele fractions using optical genome mapping alone. Citation Format: Jacob Wisotsky, Aliz Raski, Westley Sherman, Megan Roytman, Samer Al-Saffar, Jen Hauenstein, Andy Wing Chun Pang, Viren Wasnikar, Neil Miller. Comprehensive analysis of hematological malignancies with optical genome mapping and Bionano VIA™Software [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3492.

Abstract 6257: Genome wide, high-throughput, high-resolution structural variation detection at low variant allele fraction for oncology samples

Abstract Structural variation detection remains an elusive undertaking. For decades, G-banded karyotyping has remained the standard practice for nearly all hematological malignancy samples. Cancers are diseases of the genome and structural variants (SVs) are a hallmark of nearly all cancers, are often drivers of oncogenicity, and can sometimes be targeted for treatment, as highlighted by the discovery of the Philadelphia chromosome and later the development of targeted kinase inhibitors. One of the most important limitations of karyotyping, its requirement for cells to divide in culture, has prevented its application for most solid tumors and therefore, there is considerably less understanding of the impact of SVs in solid tumors. Optical genome mapping is a technique that is revolutionizing cytogenomics and unlocking the understanding of SVs in the genome at a level never seen before. It not only can detect nearly all variants that a karyotype can, but it can provide 10,000x higher resolution and does not require cell culture. Because of this, it is being adopted for oncology studies across hematological malignancies and solid tumors. This study demonstrates the performance of a new high throughput Stratys instrument for generation of optical genome map data at extraordinarily high coverage depth. With this instrument, 12 samples can be loaded at once, each loaded onto its own cartridge and the instrument can collect 400x genome coverage for each of these samples in 24 hours. The instrument has random access to the cartridges so that completed samples can be swapped out to facilitate optimal instrument run efficiency. To date, this system has been demonstrated to run 60 samples in a week, each at 400x coverage. Data from the instrument can be processed by Bionano Access™ and VIA™ software to call SVs, copy number variants (CNVs), regions of loss of heterozygosity (LOH), on a genome wide basis, and to classify those variants automatically according to guidelines such as the National Comprehensive Cancer Network (NCCN), World Health Organization (WHO), and National Health Services (NHS, United Kingdom). This new high throughput system with advanced bioinformatic data analysis has the potential to further transform structural variation analysis in cancer research. Citation Format: Alex R. Hastie, Pat Lynch. Genome wide, high-throughput, high-resolution structural variation detection at low variant allele fraction for oncology samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6257.

Abstract 2337: Accelerated optical genome mapping analysis with Stratys Compute and Guided Assembly

Abstract Background Optical genome maps (OGM) from Bionano enable the detection of genomic structural and copy number variants that cannot be detected by next-generation sequencing (NGS) technologies and are often missed by conventional cytogenetic techniques. Bionano has developed bioinformatics pipelines for calling structural and copy number variants including the Bionano Solve de novo assembly pipeline for constitutional analysis and the Rare Variant Analysis (RVA) pipeline for low-allele-fraction cancer applications. Both pipelines are computationally intensive and currently take 5-10 hours on the latest generation of the Bionano Saphyr® Compute which is deployed as a four-node compute cluster and requires significant IT resources. Methods To increase throughput and simplify the deployment of the compute resources needed to support the Stratys™ optical genome mapping instrument, Bionano has developed Stratys™ Compute. Stratys Compute improves both compute and analytical performance by adapting compute-intensive stages in the Solve pipeline to run on GPUs and by developing the Guided Assembly pipeline. Stratys Compute is powered by state-of-the-art NVIDIA RTX 6000 Ada generation cards and CUDA-optimized refinement, alignment, and structural variation detection kernels to accelerate OGM analysis. Stratys Compute is a standalone workstation placed adjacent to Stratys instrument. This will minimize the IT footprint and bypass the integration with the customer site’s data center. We have incorporated these optimizations into the new Guided Assembly pipeline, which aims to combine the low-allele fraction detection capability of the RVA pipeline with the whole genome coverage and ability to detect smaller structural variants enabled by the de novo assembly pipeline. The Guided Assembly pipeline uses the reference genome as an initial seed followed by extension, refinement, and structural variant calling. This new analysis method has been evaluated through comparison to previous results from both existing pipelines and standard benchmarking datasets used to estimate structural variant calling performance and is deployed on Stratys Compute for both constitutional and low allele-fraction applications. Results Guided Assembly has been adapted to run on GPU hardware in a simplified compute tower that can be deployed to a lab along with the Stratys instrument without the need for a dedicated server room. We found concordance between the guided assembly results and our previous de novo and RVA pipeline results. We also found increased sensitivity at low allele fractions for detecting insertion variants smaller than 5 kb and larger than 200 kb while finding equivalent performance for other variant types with the updated methods. The accelerated Guided Assembly for constitutional and low-allele-fraction applications will be available to early access customers in Q4 2023 with full commercial release in Q2 2024. Citation Format: Damla Senol Cali, Thomas Anantharaman, Martin Muggli, Samer Al-Saffar, Charles Schoonover, Neil Miller. Accelerated optical genome mapping analysis with Stratys Compute and Guided Assembly [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2337.

Abstract 2424: Detection of prostate cancer in urine by fragment-level DNA methylation analysis

Abstract Introduction: Screening for prostate cancer (PCa) with molecular markers is hindered by low tumor allele fraction (TAF) in liquid biopsy samples such as blood or urine. Others have suggested that tumor allele enrichment can be achieved by evaluating fragment-level methylation patterns generated by next generation sequencing (NGS). Here, we test this concept using target capture NGS on cfDNA from prediagnostic collected urine samples to discriminate PCa from otherwise healthy men. Methods: Buffered urines were collected from 23 patients with biopsy confirmed PCa (Gleason score 6-9) and 24 otherwise healthy age matched cancer-free controls. cfDNA was isolated and enzymatic methyl-seq libraries were prepared and enriched by hybrid capture using probes mapping to 197 differentially methylated regions (DMRs) from a previous PCa/normal tissue marker discovery study in our lab. These DMRs were subsequently vetted independently using a methylome-wide dataset from a cohort of 36 clean and disease control urines to ensure low non-cancer background signal (<1%). Indexed libraries were sequenced at 560X deduplicated coverage and fragment-level CpG methylation information was extracted from individual sample .bam files. Mapped sequencing reads exhibiting concordant methylation were used to derive a quantitative α-score. Reads had to contain at minimum 5 CpGs and have an α-value (% 5mC per read) > 60%. Positivity for the 197 markers required an α-score greater than 6 sigma (σ) above the mean of the control arm. Values were variance-adjusted to ensure equal weighting. Beta (β) analysis (mean % 5mC across all reads) of the data was also performed for comparison. Results: Of the 197 markers, 187 were positive for one or more PCa patients. Tumor-derived fragments were detected in 22 of 23 cases, which included 8 G-6, 9 G-7, 2 G-8, and 3 G-9 patients. 1 G-7 was missed, however the sequencing coverage for this sample was significantly lower than the others due to poor library enrichment. 2 of the 24 healthy controls were positive, one with extremely high fragment counts. Upon re-review of the full control cohort, this patient had been diagnosed with bladder cancer 2 years after urine collection. The 2nd patient had was diagnosed with lung cancer in a similar timeframe. The other 22 confirmed cancer-free controls had α-scores below the 6σ threshold. Using a co-captured reference gene (B3GALT6) to mimic genomic equivalents, the TAFs ranged from 0.015% to 8%. β analysis of the data detected only 13 PCa patients (>6σ) and marker positivity was lower than the fragment-level approach. Conclusion: Target capture NGS coupled with fragment-level analysis is a rational approach for sensitive and specific detection of low tumor-derived DNA in liquid biopsy samples. For PCa assay in urine, we were able to uncover positivity in cancer patients approaching 1 in 10,000 ctDNA dilution levels. These results warrant further validation studies with this and other cancers. Citation Format: Tibor Bedekovics, William R. Taylor, Zhifu Sun, Saurabh Baheti, Xiaoming Cao, Kelli N. Burger, Karen A. Doering, Patrick H. Foote, Benjamin Gochanour, Douglas, W. Mahoney, Matthew T. Gettman, John B. Kisiel. Detection of prostate cancer in urine by fragment-level DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2424.

Abstract 7335: Characterizing germline variants in patients with small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) has traditionally been considered to arise from environmental and lifestyle factors. Recent evidence has shown that germline mutations may also impact the development of SCLC, however, remains understudied. We sought to identify novel germline mutations in SCLC including unexplored copy-number variations (CNVs) in our cohort of patients. Methods: We applied a custom hybrid-capture gene panel of 191 cancer predisposition and commonly mutated genes in SCLC to a pre-treatment cohort of 67 patients with SCLC. Variant calls were performed using VarScan2 (2.3.8), Pindel (0.2.5b8) and CNVs were assessed using DeCON 1.0.2. Calls were annotated using the American College of Medical Genetics (ACMG) criteria and classified using gnomeAD (release 2.11), ESP6500 (ESP6500SI-V2), 1000Genomes, ExAC (release 0.3), COSMIC (v70), dbSNP (build 147), and ClinVar (NCBI ClinVar 20190305). We annotated variants of uncertain significance (VUS) with several in silico tools including MutationTaster, LRT, Polyphen2 HDIV, Polyphen2 HVAR, SIFT, PROVEAN, and Deepmind AlphaMissense. Kaplan-Meier survival analysis and log-rank tests were done to examine differences in overall survival (OS) and progression-free survival (PFS) from the date of diagnosis. Continuous variables were tested using Kruskal-Wallis H test and categorical variables using Fisher’s exact test. Results: The median age of our cohort was 68 years old, 64% (43/67) were male, 89% (60/67) were current/former smokers, and 58% (39/67) were Extensive-Stage. 64.6% (42/67) of patients self-reported a family history of cancer and 16% of the patients also self-reported personal history of cancer. In terms of self-reported ancestral background, 76% of were Caucasian, 1.5% were Middle-Eastern, 3% were Latin American, 14% were Asian, and 4.5% were Black. We identified pathogenic/likely pathogenic alterations in 7/67 patients. Five (71%) were novel alterations (BCORL1, FANCC, ATR, and BBC3) and a novel CNV (SLFN11) with the remaining two (29%) previously described mutations (CHEK1 and BRIP1). We identified 191 VUS in 60/67 patients. 4.7-14.1% of VUS were in silico predicted to be pathogenic. Patients with the 7 pathogenic alterations had a numerically larger OS (HR = 0.50, 95%CI = 0.18-1.39, p = 0.18) and PFS (HR = 0.45, 95%CI = 0.16 - 1.24, p = 0.11) compared to the rest of the cohort. We identified 20 variants at low variant allele fractions (0.05 to 0.24), likely somatic mutations. Some genes identified are implicated with clonal hematopoiesis such as GNAS, STAG, TET2, and MGA. Conclusion: In summary, we have identified several novel germline alterations (mutations and CNVs) in patients with SCLC. In addition, we utilized in silico prediction models to categorize potential high-risk VUS. Our findings suggest that beyond tobacco exposure germline alterations may also modulate development of tumorigenesis in SCLC patients. Citation Format: Sami Ul Haq, Gregory Downs, Luna Jia Zhan, Danielle Sacdalan, Janice J. Li, Vivek Philip, Raymond H. Kim, Geoffrey Liu, Scott V. Bratman, Peter J. Sabatini, Benjamin H. Lok. Characterizing germline variants in patients with small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7335.

ArCH: improving the performance of clonal hematopoiesis variant calling and interpretation.

The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion, termed clonal hematopoiesis (CH), is associated with increased risk of hematologic malignancies and other adverse outcomes. CH is generally present at low allelic fractions, but clonal expansion and acquisition of additional mutations leads to hematologic cancers in a small proportion of individuals. With high depth and high sensitivity sequencing, CH can be detected in most adults and its clonal trajectory mapped over time. However, accurate CH variant calling is challenging due to the difficulty in distinguishing low frequency CH mutations from sequencing artifacts. The lack of well-validated bioinformatic pipelines for CH calling may contribute to lack of reproducibility in studies of CH. Here, we developed ArCH, an Artifact filtering Clonal Hematopoiesis variant calling pipeline for detecting single nucleotide variants and short insertions/deletions by combining the output of four variant calling tools and filtering based on variant characteristics and sequencing error rate estimation. ArCH is an end-to-end cloud-based pipeline optimized to accept a variety of inputs with customizable parameters adaptable to multiple sequencing technologies, research questions, and datasets. Using deep targeted sequencing data generated from six acute myeloid leukemia patient tumor: normal dilutions, 31 blood samples with orthogonal validation, and 26 blood samples with technical replicates, we show that ArCH improves the sensitivity and positive predictive value of CH variant detection at low allele frequencies compared to standard application of commonly used variant calling approaches. The code for this workflow is available at: https://github.com/kbolton-lab/ArCH.

Somatic PIK3CA Variants Are Associated With Eccrine Angiomatous Hamartomas

Objectives: Eccrine angiomatous hamartoma (EAH) is a rare vascular anomaly with mixed eccrine and vascular components, typically identified in children. While benign, EAH can cause significant morbidity and be difficult to treat. The aims of this case series were to identify all patients with EAH that have been seen at the Queensland Children’s Hospital and describe their phenotypic and somatic genotypic details, in an effort to contribute to the limiting understanding and literature surrounding this condition. Methods: Individuals with EAH were retrospectively identified through engagement in a multidisciplinary vascular anomaly clinic in a tertiary Australian children’s hospital. All individuals had a previous histological diagnosis of EAH. High-read-depth sequencing of a panel of 27 genes known to be associated with vascular anomalies was undertaken on affected tissue. Samples were rereviewed by a senior pathologist and geneticist for this study. Results: Five cases of EAH were identified. All were associated with 1 of 3 somatic PIK3CA variants (c.1633G>A;p.Glu545Lys, c.1624G>A;p.Glu542Lys, and c.3140A>G;p.Histo1047Arg) in low allele fractions. These variants have previously been reported in a range of tumors and vascular anomalies, including PIK3CA-related overgrowth spectrum, but not in EAH. Conclusion: Occurrence of somatic PIK3CA variants in EAH provides evidence for a novel gene-disease association and is plausibly the cause of EAH in some individuals. This finding expands the phenotypic spectrum of PIK3CA, contributes to understanding of the pathophysiology of this rare condition, and may avail molecularly targeted therapy in the future.

Molecular characterization of 13 patients with PIK3CA-related overgrowth spectrum using a targeted deep sequencing approach.

Activating variants in the PIK3CA gene cause a heterogeneous spectrum of disorders that involve congenital or early-onset segmental/focal overgrowth, now referred to as PIK3CA-related overgrowth spectrum (PROS). Historically, the clinical diagnoses of patients with PROS included a range of distinct syndromes, including CLOVES syndrome, dysplastic megalencephaly, hemimegalencephaly, focal cortical dysplasia, Klippel-Trenaunay syndrome, CLAPO syndrome, fibroadipose hyperplasia or overgrowth, hemihyperplasia multiple lipomatosis, and megalencephaly capillary malformation-polymicrogyria (MCAP) syndrome. MCAP is a sporadic overgrowth disorder that exhibits core features of progressive megalencephaly, vascular malformations, distal limb malformations, cortical brain malformations, and connective tissue dysplasia. In 2012, our research group contributed to the identification of predominantly mosaic, gain-of-function variants in PIK3CA as an underlying genetic cause of the syndrome. Mosaic variants are technically more difficult to detect and require implementation of more sensitive sequencing technologies and less stringent variant calling algorithms. In this study, we demonstrated the utility of deep sequencing using the Illumina TruSight Oncology 500 (TSO500) sequencing panel in identifying variants with low allele fractions in a series of patients with PROS and suspected mosaicism: pathogenic, mosaic PIK3CA variants were identified in all 13 individuals, including 6 positive controls. This study highlights the importance of screening for low-level mosaic variants in PROS patients. The use of targeted panels with deep sequencing in clinical genetic testing laboratories would improve diagnostic yield and accuracy within this patient population.

TP53 mutations and Their Impact on Survival in Patients with Myeloproliferative Neoplasms

Introduction: TP53 mutations in patients with myeloproliferative neoplasms (MPN) are associated with poor prognosis, including progression to blast phase MPN. However, low variant allele fraction (VAF) TP53 mutations have been reported to remain stable over years in chronic phase MPN. A major unmet clinical need in MPN is the ability to discriminate patients with TP53-mutant MPN who are at high-risk of secondary AML (sAML) and warrant immediate intervention from those who are at lower risk of sAML in whom active surveillance can be employed. Therefore, we sought to identify parameters associated with leukemic transformation and overall survival in the context of MPN with genetic aberrations in TP53. Materials and Methods: We retrospectively analyzed a cohort of 947 MPN patients from the Dana-Farber Cancer Institute Hematologic Malignancies Data Repository (HMDR) with at least one clinical next-generation sequencing (NGS) panel performed. Patient characteristics such as age at MPN diagnosis, gender, MPN subtype and driver mutations were recorded. Furthermore, information about the course of disease was extracted including occurrence of sAML and overall survival (Figure 1). We also analyzed type and number of additional mutations as well as cytogenetics. With respect to TP53-specific parameters, we evaluated the number of TP53 mutations, TP53 VAF, loss of heterozygosity (LOH) at the TP53 locus, phenotypic annotations of TP53 (i.e. PHANTM score) and 17p deletion. We defined “multi-hit” TP53 as the presence of two or more TP53 mutations, TP53 VAF higher than 50%, TP53 mutation plus 17p deletionor TP53 mutation and documented LOH. Results: A total of 947 patients were analyzed, of which 40 harbored at least one detectable TP53 mutation. A total of 13 patients were found to have a multi-hit TP53 mutations defined by > 50% VAF in 6 patients, two or more TP53 mutations in 5 patients and TP53 mutation + 17p deletion in 5 patients. The MPN diagnosis at time of TP53 mutation detection was post ET/PV myelofibrosis (secondary MF) (n=23, 58%), primary myelofibrosis (MF) (n=7, 18%), pre-fibrotic MF (n=2, 5%), essential thrombocythemia (ET) (n=6, 15%) and polycythemia vera (PV) (n=2, 5%). Two patients with ET and one patient with PV did not have a concurrent in-house bone marrow biopsy performed at the time the TP53 mutation was detected. Two patients with ET developed sAML within 12 months of TP53 mutation detection, without prior mention of fibrosis. Age at first MPN diagnosis was not significantly different between patients with or without TP53 mutation. The average time from initial MPN diagnosis to detection of the first TP53 mutation was 9 years (range: 0-33 years). The most common MPN driver mutation among patients with TP53 mutations was JAK2 (75%), followed by CALR (13%)and MPL (5%). Out of all TP53-mutated patients, 8% showed a triple negative status. The most frequent additional mutations among patients with TP53 mutations were TET2 (25%), U2AF1 (15%), ASXL1 (13%), and DNMT3A (10%). There was no significant difference between single-hit and multi-hit TP53 status regarding MPN subtype, driver mutations and co-mutations (Table 1). Seven patients (single-hit: 15%, multi-hit: 23%) with a TP53 mutation developed sAML during the course of their disease, compared with only 3% of all patients without a TP53 mutation and 50% (single-hit: 41%, multi-hit: 69%) were deceased at the time of the last follow-up compared to 18% of all patients without a TP53 mutation. We focused on overall survival from the initial MPN diagnosis and considered whether patients developed bone marrow fibrosis during their disease course (Figure 1). Survival did not differ significantly between single-hit TP53 and patients with multi-hit TP53 (p=0.2), but survival did differ significantly between multi-hit TP53 patients and TP53 wildtype patients with MF/prefibrotic MF/Secondary MF (p=0.02) as well as compared to all MPN patients without a TP53 mutation (p<0.001). Survival was not significantly different between single-hit TP53 and TP53 wildtype MF/prefibrotic MF/Secondary MF patients (p=0.4). Conclusions: In a large cohort of 947 molecularly characterized MPN patients, 4% of the cohort developed a TP53 mutation during their course of disease. 18% of all TP53-mutant patients developed sAML with an adverse effect on overall survival for patients with multi-hit but not single-hit TP53 mutations.

LONGITUDINAL WGS AND WES OF GLIOBLASTOMA REVEAL POTENTIAL PATHWAY TARGETS

Abstract AIMS Treating glioblastoma (GBM) patients remains challenging as the current regimens result in the recurrence of a treatment-resistant and fatal tumour. We aimed to identify candidate pathways that, when mutated, confer treatment resistance. Targeting these pathways may improve GBM treatment effcacy. METHOD We performed WGS and WES on 46 matched primary and recurrent GBMs (the Discovery cohort) and used data from additional 188 samples from the Glioma Longitudinal AnalySiS consortium (GLASS: Validation cohort). First, we identified variants that were shared between primary and recurrent GBMs. We then highlighted those most likely to be involved in treatment resistance, i.e. with: 1) low allele fractions at primary and high in recurrent, 2) high allele fractions in both primary and recurrent. Finally, we performed enrichment analysis using these variants to identify pathways that may drive clonal expansion from primary to recurrent. RESULTS The analysis of variants that increased from primary to recurrent revealed three significant disrupted path- ways in the Discovery cohort; “Activation of BH3 Only Proteins” (Reactome), “P53 pathway” (BIOCARTA), and “Intrinsic Pathway for Apoptosis” (REACTOME). Six distinct genes caused the aberration of these pathways. The same pathways were significantly disrupted in the recurrent variants group of the Validation cohort but were solely caused by TP53 alterations. In the Discovery cohort, “Activation of BH3 Only Proteins” was altered in 24% of the patients. CONCLUSIONS Investigating how pathway mutations alter in GBM through treatment has led to identifying potential candidate mechanisms to be further studied in the laboratory.

Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers.

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5' unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify areference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine.

Highly Sensitive Enrichment of Low-Frequency Variants by Hairpin Competition Amplification.

Gene mutations are inevitably accumulated in cells of the human body. It is of great significance to detect mutations at the earliest possible time in physiological and pathological processes. However, genotyping low-copy tumor DNA (ctDNA) in patients is challenging due to abundant wild DNA backgrounds. One novel strategy to enrich rare mutations at low variant allele fractions (VAFs) with quantitative polymerase chain reaction (qPCR) and Sanger sequencing was contrived by introducing artificial hairpins into amplicons to compete with primers, coined as the hairpin competition amplification (HCA) system. The influence imposed by artificial hairpins on primer-binding in a high-temperature PCR system was investigated for the first time in this work, paving the way for the optimization of HCA. HCA differs from the previously reported work in which hairpins are formed to inhibit extension of wild-type DNA using 5-exonuclease-negative polymerase, where the readout is dependent on melting curve analysis after asymmetric PCR. Targeted at six different variants, HCA qPCR and HCA Sanger-enriched mutant DNA at VAFs as low as 0.1 or 0.01% were performed. HCA demonstrated advantages in multiplex reaction and temperature robustness. In profiling gene status from 12 lung cancer ctDNA samples and 16 thyroid cancer FNA DNA samples, HCA demonstrated a 100% concordance rate compared to ddPCR and commercial ARMS kit. HCA qPCR and Sanger sequencing can enrich low-abundance variants with high sensitivity and temperature robustness, presenting a novel and effective tool for precision diagnosis and treatment of rare variant diseases.

Combined Tumor-Based BRCA1/2 and TP53 Mutation Testing in Ovarian Cancer.

Somatic/germline BRCA1/2 mutations (m)/(likely) pathogenic variants (PV) (s/gBRCAm) remain the best predictive biomarker for PARP inhibitor efficacy. As >95% of high-grade serous ovarian cancers (HGSOC) have a somatic TP53m, combined tumor-based BRCA1/2 (tBRCA) and TP53 mutation testing (tBRCA/TP53m) may improve the quality of results in somatic BRCAm identification and interpretation of the 'second hit' event, i.e., loss of heterozygosity (LOH). A total of 237 patients with HGSOC underwent tBRCA/TP53m testing. The ratio of allelic fractions (AFs) for tBRCA/TP53m was calculated to estimate the proportion of cells carrying BRCAm and to infer LOH. Among the 142/237 gBRCA results, 16.2% demonstrated a pathogenic/deleterious variant (DEL) gBRCA1/2m. Among the 195 contributive tumor samples, 43 DEL of tBRCAm (22.1%) were identified (23 gBRCAm and 20 sBRCAm) with LOH identified in 37/41 conclusive samples. The median AF of TP53m was 0.52 (0.01-0.93), confirming huge variability in tumor cellularity. Initially, three samples were considered as wild type with <10% cellularity. However, additional testing detected a very low AF (<0.05) in both BRCA1/2m and TP53m, thus reidentifying them as sBRCA1/2m. Combined tBRCA/TP53m testing is rapid, sensitive, and identifies somatic and germline BRCA1/2m. AF TP53m is essential for interpreting sBRCA1/2m in low-cellularity samples and provides indirect evidence for LOH as the 'second hit' of BRCA1/2-related tumorigenesis.

Analytical performance assessment of ASPYRE-Lung: A new assay for rapid detection of actionable variants from tissue and plasma in NSCLC patients.

e15038 Background: We developed ASPYRE technology to make detection of genomic variants in oncology patients more accessible. Although ASPYRE is not traditional PCR, it can be performed using standard qPCR instruments on nucleic acid extracted from FFPE tissue or plasma and is therefore easily adoptable by clinical laboratories. Using ASPYRE, we designed a targeted panel called ASPYRE-LungTM consisting of 114 variants in 11 genes ( ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET &amp; NTRK1/2/3) to inform the clinical management of patients with NSCLC. We have previously published general assay descriptions of ASPYRE-Lung (Silva et al., 2021, Scientific Reports and Gray et al., 2022, BMC Medical Genomics). Here we extend our prior studies with a detailed analytic performance assessment across all variants using precisely controlled synthetic samples. We determined assay-wide performance using samples with low variant allele fractions (VAFs) near the limit of detection (LoD) of the assay and compared results across 2 separate laboratory settings. Methods: Nine independent batches of reagents were split between 2 laboratories. Contrived test samples were prepared with known VAFs (DNA variants) or copy numbers (RNA variants). Each sample contained a single variant with VAFs of 0.1-0.5% for DNA variants, and 6-150 copies for RNA variants. Multiple replicates were performed at both testing sites using 20ng DNA/6ng RNA. Data was processed using an internally designed algorithm and analyzed using a machine learning classifier which determined the presence or absence of each individual variant. Results: Study data resulted in a total of 133,083 variant calls from 2,319 samples. For each variant at each VAF or copy number, the detection rate was calculated, and the results for each variant were used to calculate an LoD95 using probit regression. Target assay specifications and the assay-wide analytical performance data are shown in the table below. Conclusions: The data show that ASPYRE-Lung meets or exceeds target specifications for the detection of single nucleotide variants (SNVs), insertions/deletions (indels), and gene fusions. Estimates of LoD for key variants fall within target specifications, and assay-wide performance demonstrates highly sensitive detection of each class of variant. Performance was similar at the two laboratory sites further supporting the clinical potential of the assay. Moreover, the assay only requires 20ng DNA and as little as 6ng RNA, depending on sample type, so it is highly suitable for use with typical NSCLC patient samples such as needle core biopsies or plasma where available nucleic acid may be limited. This data supports further clinical development of ASPYRE-Lung. [Table: see text]

Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05-0.5% within 2h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.

Genetic events associated with venetoclax resistance in CLL identified by whole-exome sequencing of patient samples

AbstractAlthough BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the combination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-κB gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.