Detection Of Single Nucleotide Variants Research Articles

Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas: a proof-of-concept study.

Analyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA). In 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up. A total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse. In summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.

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]

Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a.

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30–40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.

Temperature-boosted PAM-less activation of CRISPR-Cas12a combined with selective inhibitors enhances detection of SNVs with VAFs below 0.01%

The precise identification of rare single nucleotide variations (SNVs) concomitant with excess wild-type DNA is a valuable method for minimally invasive disease diagnosis and early prediction of drug responsiveness. Selective enrichment of mutant variants via strand displacement reaction offers an ideal approach of SNVs analysis but fails to differentiate wildtype from mutants with variant allele fraction (VAF) < 0.01%. Here, we demonstrate that integration of PAM-less CRISPR-Cas12a and adjacent mutation-enhanced inhibition of wild-type alleles enables highly sensitive measurement of SNVs well below the 0.01% VAF threshold. Raising the reaction temperature to the upper limit of LbaCas12a helps to boost PAM-less activation of collateral DNase activity, which can be further enhanced using PCR additives, leading to ideal discriminative performance for single point mutations. Along with selective inhibitors bearing additional adjacent mutation, it allowed detection of model EGFR L858R mutants down to 0.001% with high sensitivity and specificity. Preliminary investigation on adulterated genomic samples prepared in two different ways also suggests that it can accurately measure ultralow-abundance SNVs extracted directly from clinical samples. We believe that our design, which combines the superior SNV enrichment capability of strand displacement reaction and unparalleled programmability of CRISPR-Cas12a, has the potential to significantly advance current SNV profiling technologies.

Abstract 3553: Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes

Abstract Introduction: Retinoblastoma (Rb) is the most common childhood intraocular cancer. Tissue biopsy of Rb can cause tumor spread, so it is contraindicated. We demonstrated that aqueous humor (AH), an ocular fluid, is a high-yield liquid biopsy enabling in vivo detection of tumor-derived cell-free DNA (cfDNA) thus overcoming the contraindication of biopsy. Somatic genomic alterations, including both somatic copy number alterations (SCNAs) and single nucleotide variations (SNVs) on RB1 gene, have been able to be identified in the same clinical samples but with two separate sequencing runs. In this study, we first developed a single targeted sequencing method to identify both SCNAs and RB1 SNVs. With this method, we further investigated the degree of genomic concordance between paired tumor and AH samples from the same Rb eye. Materials and Methods: 11 paired AH and Rb tumor samples were included in the study. cfDNA of AH and tumor DNA of enucleated Rb eyes were isolated with QIAgen commercial kits. DNAs were constructed into whole genome libraries followed by hybridization target enrichment (Agilent SureSelect). The enrichment assay covers the whole length of RB1 and MycN genes, the all-exon regions of BCOR and CREBBP genes, and a whole genome CNV backbone. Libraries were submitted to Illumina HiSeq 4000 platform for fastq data generation with 2 × 150bp mode. The bioinformatics pipeline was optimized to generate SCNA profiles from targeted sequencing reads, along with SNV calling. Results: For SCNA profiles, 11/11 AH samples (100%) and 8/11 tumor samples (72.72%) have positive RB-SCNA signatures. Strong concordances were observed between AH and tumor SCNA profiles (median = 90.1%) with targeted sequencing reads. In total, 9 disease-driving RB1 SNVs were identified in 6 patients (54.5%). 7/9 (77.8%) of the variants were shared between AH and tumor samples, while the AH and tumor each contained one unique SNV. In all SNVs, the AH displayed a higher allele frequency. Notably, 4/11 samples have focal RB1 gene deletion detected with SCNA profiling, which may explain the difficulties of RB1 SNV detection in some samples. Further, one case was driven by a MYCN gene amplification with no RB1 alterations. Conclusions: This study presented highlights the utility of a single method to call both SVNs and SCNAs in a single clinical sample, with enriched tumor information detected in AH compared with tumor. This study further strengthens the utility of AH as a liquid biopsy platform for Rb eyes. Citation Format: Liya Xu, Mike J. Schmidt, Rishvanth K. Prakabar, Peter Kuhn, James Hicks, Jesse Berry. Simultaneous somatic copy number alterations and single nucleotide variants detection in paired aqueous humor and tumors from retinoblastoma eyes. [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 3553.

Abstract 5597: Enhanced detection and classification of cell-free DNA alterations through matched normal analyses with PGDx elioTM plasma complete

Abstract Liquid biopsies represent a transformation in the management of cancer as they have the potential to detect, characterize, and monitor cancers earlier than can be achieved with conventional diagnostic modalities. However, cell-free DNA (cfDNA)-based alterations can be derived from the tumor, germline, or may be associated with clonal hematopoiesis (CH), which can confound non-invasive tumor profiling, molecular response assessment, and clonal evolution analyses through inaccurate variant classification. To facilitate global access to a decentralized liquid biopsy solution to address this, we developed and validated the 521 gene PGDx elio plasma complete test for paired analysis of cfDNA and matched leukocyte DNA. PGDx elio plasma complete enables detection of single nucleotide variants, insertions and deletions, copy number amplifications, translocations, microsatellite instability, blood tumor mutation burden, and loss of heterozygosity. We first optimized the assay workflow to incorporate genomic DNA derived from leukocytes to facilitate direct detection and characterization of germline alterations as well as those that may be associated with CH, resulting in de-duplicated, error-corrected sequencing coverage of approximately 1,750-fold. A fully automated bioinformatics algorithm was then developed and validated to perform integrated analyses of cfDNA-derived alterations to assign the appropriate biological source of these variants. To assess the impact of these paired sample analyses, we analyzed the blood samples obtained from 24 patients representing seven different solid tumor types (breast, colorectal, gastric, gastro-esophageal junction, lung, and melanoma). Across this cohort, of the alterations detected in cfDNA (n=322), 87.3% were correctly classified as somatic, germline or CH without the patient-matched normal blood sample. Specifically, of the variants that were determined to be associated with CH (n=26), only 35% were appropriately assigned without the paired comparison. Additional sources of discordance for somatic and germline alterations were primarily attributed to patients with high levels of ctDNA where differentiation of these variant sources can be challenging through solely computational-based techniques. Taken together, these data demonstrate that through the integrated analysis of cell-free DNA and matched leukocyte DNA, classification of the source of cfDNA-derived alterations can be achieved, which may improve the accuracy of non-invasive tumor profiling, molecular response assessment, and clonal evolution analyses. Citation Format: Tonya N. Watkins, Paul McGregor, David Riley, Tolga Ayazseven, Kristy Waskiewicz, Kelly M. Gerding, Ellen L. Verner, Amy Greer, Aanavi Karandikar, Rami Zahr, Kenneth C. Valkenburg, Jamie Platt, Samuel V. Angiuoli, Mark Sausen. Enhanced detection and classification of cell-free DNA alterations through matched normal analyses with PGDx elioTM plasma complete. [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 5597.

Abstract 6687: Prevalence of four major gene mutation classes in a survey of ctDNA recovery from early and advanced stage NSCLC

Abstract Introduction: Circulating tumor DNA (ctDNA) has emerged as a powerful biomarker with many applications in oncology including early and late-stage disease prognosis and in treatment decision making. Liquid biopsies have become critical in non-small cell lung cancer (NSCLC) given the patient risks associated with biopsies. It is well established that circulating nucleic acid levels representative of all solid tumors are increased in advanced stages of the disease. Less well characterized is the status of cfDNA levels in the earliest stages of solid tumor progression. We used validated methods for quantitative fluorometry and a high sensitivity amplicon based NGS method to survey cfDNA recovery and prevalence for the four major mutation classes (copy number amplifications (CNAs), single nucleotide variations (SNVs), fusions, and insertions/deletions (INDELs)) from patients previously diagnosed with NSCLC. Methods: Cell-free nucleic acid was extracted from 1,139 late-stage and 185 early-stage NSCLC plasma specimens. cfDNA was quantified using fluorometry, and the 52 gene GeneStrat NGS Test was used for the detection of CNAs, SNVs, fusions and INDELs. We evaluated cfDNA concentration (normalized to plasma input volume), quantity in circulation, and the prevalence of the four classes of mutations in early-stage (I-IIIA) versus late-stage (IIIB-IV) NSCLC. Results: We observed significantly lower average concentrations of cf nucleic acids in plasma recovered from early versus late-stage NSCLC specimens (early-stage: 13.7 ng/mL; range (3.14, 118.65); n = 185; late-stage: 18.3 ng/mL; range (0.67, 187.2); n =1,139; p=0.00176). We also evaluated total cfDNA recovered for the sample set and observed significantly lower average measurements for early-stage specimens versus late-stage (early-stage: 101.4 ng; range (18.38, 652.6); n=185; late-stage: 140.5 ng; range (9.802, 1534); n=1,139; p=0.00692). We do note that there were overlaps at the low end of the ranges. Also significant was the finding that all mutation classes were more prevalent in late versus early-stage NSCLC. Specifically, 56 of 58 CNAs (96.6%); 737 of 805 SNVs (91.6%); 42 of 43 INDELs (97.3%) and 12 fusions (100%) were associated with late-stage specimens. Conclusion: In this observational study cohort, cfDNA yields were significantly higher in late-stage NSCLC patients compared to early-stage. We also observed significantly more and varied mutations in plasma from late-stage patients. These initial findings need to be extended to additional cancers and refined by stage at diagnosis. We are currently expanding our study to include additional technologies and panels to assess ctDNA mutations especially for the early-stage patients. Quantitative cfDNA data may be of utility in diagnosis, monitoring and recurrence of cancer. Citation Format: Claire Gould, Leisa Jackson, Colin Cochran, Audrey Audetat, Brittany D'Alessio, Amanda Weaver, Janice Riley, Gary A. Pestano. Prevalence of four major gene mutation classes in a survey of ctDNA recovery from early and advanced stage NSCLC [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 6687.

Abstract 6220: Development of a mutation profiling NGS assay to facilitate clinical decisions in hematologic malignancies

Abstract Introduction: Accurate diagnosis, prognosis and treatment of hematologic disorders from both myeloid and lymphoid origin requires assessing somatic mutation status in an increasingly large number of genes. Here we present the results of the assay development and validation of a targeted next generation sequencing panel which provides full exon sequencing of 141 genes associated with myeloid and lymphoid malignancies, as well as the ability to detect clinically relevant copy number alterations (CNAs) and certain sub-gene level CNAs. Methods: Based on review of clinical guidelines, 141 genes including 72 myeloid and 84 lymphoid associated genes were selected. The assay interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The assay also identifies CNAs in 16 genes, FLT3 internal tandem duplicates (ITDs), and select non-coding pathogenic variants. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood or bone marrow followed by sequencing on Illumina sequencers. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/Indels or digital multiplexed ligation-dependent probe amplification for CNAs. In addition, analytical sensitivity was assessed using cell line dilution series and specificity by reference samples from Genome in a Bottle (GIAB). Small indels were defined as &amp;lt;25bp while long indels are ≥ 25bp. Results: Interim estimates of positive percentage agreement (PPA) and specificity have been determined for SNV/Indels, FLT3-ITDs, and CNVs, with further testing ongoing. PPA of SNVs was 100% (218/218 SNVs; n = 134 samples). PPA was also 100% for both small indels (21/21; n = 14 samples) and long indels (6/6; n = 6 samples). A dilution series of 8 positive clinical samples at 4 different dilutions showed SNV and small indel detection of 100% at allele frequency (AF) ≥ 3%. Long indel detection was 87.5% at AF ≥ 5%. FLT3-ITD was evaluated with 2 cell line samples at 5 different dilutions with 100% detection at AF ≥ 5%. PPA of CNAs were 95.7% (157/164) in 46 clinical samples. CNA detection was also evaluated with 8 cell line samples at 4 different dilutions showing 92.3% detection at a copy number ratio threshold ± 0.15. A preliminary estimate of specificity was determined using 4 replicates of the GIAB sample, NA12878, and was &amp;gt;99.99% for SNVs and small indels (n=6.9 × 105). Finally, a model for gender classification was created using a training cohort of 70 clinical samples. The performance was evaluated on a test set of 144 clinical samples with 100% PPA (144/144). Conclusions: Taken together, these data demonstrate that the developed NGS assay is a sensitive, specific, and accurate assay with high clinical utility to inform treatment decision making in hematologic malignancies. Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Stan Letovsky, Li Cai, Eric A. Severson, Shakti Ramkissoon, Anjen Chenn, Marcia Eisenberg, Eyad Almasri, Taylor Jensen. Development of a mutation profiling NGS assay to facilitate clinical decisions in hematologic malignancies [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 6220.

Noninvasive genomic profiling of somatic mutations in oral cavity cancers

ObjectivesSomatic mutations may predict prognosis, therapeutic response, or cancer progression. We evaluated targeted sequencing of oral rinse samples (ORS) for non-invasive mutational profiling of oral squamous cell carcinomas (OSCC). Materials and methodsA custom hybrid capture panel targeting 42 frequently mutated genes in OSCC was used to identify DNA sequence variants in matched ORS and fresh-frozen tumors from 120 newly-diagnosed patients. Receiver operating characteristic (ROC) curves determined the optimal variant allele fraction (VAF) cutoff for variant discrimination in ORS. Behavioral, clinical, and analytical factors were evaluated for impacts on assay performance. ResultsHalf of tumors involved oral tongue (50 %), and a majority were T1-T2 tumor stage (55 %). Median depth of sequencing coverage was 260X for OSCC and 1,563X for ORS. Frequencies of single nucleotide variants (SNVs) at highly mutated genes (including TP53, FAT1, HRAS, NOTCH1, CDKN2A, CASP8, NFE2L2, and PIK3CA) in OSCC were highly correlated with TCGA data (R = 0.96, p = 2.5E−22). An ROC curve with area-under-the-curve (AUC) of 0.80 showed that, at an optimal VAF cutoff of 0.10 %, ORS provided 76 % sensitivity, 96 % specificity, but precision of only 2.6E−4. At this VAF cutoff, 206 of 270 SNVs in OSCC were detected in matched ORS. Sensitivity varied by patient, T stage and target gene. Neither downsampled ORS as matched control nor a naïve Bayesian classifier adjusting for sequencing bias appreciably improved assay performance. ConclusionTargeted sequencing of ORS provides moderate assay performance for noninvasive detection of SNVs in OSCC. Our findings strongly rationalize further clinical and laboratory optimization of this assay, including strategies to improve precision.

Abstract P2-26-06: Association between CK+/CD45+ circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) alterations in advanced breast cancer patients

Abstract Background: Circulating tumor cells (CTCs) in breast cancer (BC) are commonly defined as epithelial cells (EPCAM and cytokeratin (CK) positive), lacking the universal blood cell marker CD45. Nonetheless, CTCs expressing both CK and CD45 (= dual-positive, DP cells) can be observed in the blood of cancer patients. Early evidence suggests that DP cells might derive from the fusion of tumor cells and macrophages, and we have previously demonstrated that they present aberrant genomes and are associated with worse prognosis in BC [1,2]. Here, to further investigate the mechanisms/pathways underlying their presence, we analyzed the association between DP cells and circulating tumor DNA (ctDNA) alterations. Methods: Blood samples were collected from patients with advanced BC (aBC), before starting a new line of therapy. All patients were enrolled in a prospective clinical trial. For CTC and DP cells analysis, 7.5 ml of blood collected in CellSave® tubes was processed with the FDA-approved CellSearch® platform (positivity cutoffs were ≥1 cell for DPcells and ≥5 cells for CTCs). For ctDNA analysis, plasma was collected from Streck stabilizing tubes and analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs), insertions/deletions (indels), gene fusions/rearrangements and copy number variations (CNVs), which were then classified into pathways based on previously defined profiles generated on the Cancer Genome Atlas database (RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Associations between ctDNA-detected gene alterations and circulating cell types were analyzed through chi square test, while mutant allele frequency (MAF) and number of detected alterations (NDA) were tested by Mann Whitney test. Results: We analyzed blood samples from 169 patients with luminal-like (n=80), HER2+ (n=34) and triple-negative (n=52) aBC. DPcells were detected in 85 patients (50.3 %, range 0-53), of which 40 (47 %) were CTC-positive and 45 (53%) CTC-negative. Somatic ctDNA alterations were detected in all analyzed samples. In the overall population, the presence of ≥1 DPcell was associated with SNVs in the cell cycle pathway (p = 0.043), a numerically higher incidence was also observed for CNVs in this pathway. SNVs and CNVs in the cell cycle pathway were associated with CTCs ≥ 5 as well (p = 0.005 and p = 0.003, respectively). Moreover, associations with CTCs ≥ 5 were observed for RTK SNVs and CNVs (p = 0.041 and p = 0.046, respectively), PI3K SNVs and CNVs (p = 0.006 and p = 0.007, respectively), MYC SNVs and CNVs (p = 0.042). No associations were observed in terms of MAF and NDA. In the luminal-like subgroup an association was highlighted for CNVs in the cell cycle pathway, p = 0.038. CTCs ≥ 5 were associated with PI3K SNVs (p = 0.031). In the triple-negative subgroup DPcells were associated with SNVs in the RAF pathway (p = 0.041), whereas CTCs ≥ 5 were associated with PI3K SNVs and CNVs (p = 0.044 and p = 0.024, respectively) and RTK SNVs (p = 0.008). In the HER2 positive subgroup, a higher MAF and number of detected SNVs was observed for samples with ≥1 DPcell (p = 0.0286 and p = 0.0099, respectively). Conclusions: The study analyzed ctDNA features associated with canonical and CK+/CD45+ CTCs, showing differential gene alteration profiles. Cell cycle pathway SNVs were common in both CTC populations, while other pathways (RTK, PI3K, MYC and RAF) were significantly altered in a mutually exclusive pattern. These results suggest that DPcells might have a different biological meaning compared to canonical CTCs. More studies need to be conducted to better characterize this understudied CTC subpopulation and understand their specific contribution to cancer progression.

Analytical validation and implementation of a pan cancer next-generation sequencing panel, CANSeqTMKids for molecular profiling of childhood malignancies.

Next-Generation Sequencing (NGS) allows rapid analysis of multiple genes for the detection of clinically actionable variants. This study reports the analytical validation of a targeted pan cancer NGS panel CANSeqTMKids for molecular profiling of childhood malignancies. Analytical validation included DNA and RNA extracted from de-identified clinical specimens including formalin fixed paraffin embedded (FFPE) tissue, bone marrow and whole blood as well as commercially available reference materials. The DNA component of the panel evaluates 130 genes for the detection of single nucleotide variants (SNVs), Insertion and Deletions (INDELs), and 91 genes for fusion variants associated with childhood malignancies. Conditions were optimized to use as low as 20% neoplastic content with 5ng of nucleic acid input. Evaluation of the data determined greater than 99% accuracy, sensitivity, repeatability, and reproducibility. The limit of detection was established to be 5% allele fraction for SNVs and INDELs, 5 copies for gene amplifications and 1,100 reads for gene fusions. Assay efficiency was improved by automation of library preparation. In conclusion, the CANSeqTMKids allows for the comprehensive molecular profiling of childhood malignancies from different specimen sources with high quality and fast turnaround time.

Targeted High-Throughput Sequencing Enables the Detection of Single Nucleotide Variations in CRISPR/Cas9 Gene-Edited Organisms.

Similar to genetically modified organisms (GMOs) produced by classical genetic engineering, gene-edited (GE) organisms and their derived food/feed products commercialized on the European Union market fall within the scope of European Union Directive 2001/18/EC. Consequently, their control in the food/feed chain by GMO enforcement laboratories is required by the competent authorities to guarantee food/feed safety and traceability (2003/1829/EC; 2003/1830/EC). However, their detection is potentially challenging at both the analytical and interpretation levels since this requires methodological approaches that can target and detect a specific single nucleotide variation (SNV) introduced into a GE organism. In this study, we propose a targeted high-throughput sequencing approach, including (i) a prior PCR-based enrichment step to amplify regions of interest, (ii) a sequencing step, and (iii) a data analysis methodology to identify SNVs of interest. To investigate if the performance of this targeted high-throughput sequencing approach is compatible with the performance criteria used in the GMO detection field, several samples containing different percentages of a GE rice line carrying a single adenosine insertion in OsMADS26 were prepared and analyzed. The SNV of interest in samples containing the GE rice line could successfully be detected, both at high and low percentages. No impact related to food processing or to the presence of other crop species was observed. The present proof-of-concept study has allowed us to deliver the first experimental-based evidence indicating that the proposed targeted high-throughput sequencing approach may constitute, in the future, a specific and sensitive tool to support the safety and traceability of the food/feed chain regarding GE plants carrying SNVs.

Control-independent mosaic single nucleotide variant detection with DeepMosaic.

Mosaic variants (MVs) reflect mutagenic processes during embryonic development and environmental exposure, accumulate with aging and underlie diseases such as cancer and autism. The detection of noncancer MVs has been computationally challenging due to the sparse representation of nonclonally expanded MVs. Here we present DeepMosaic, combining an image-based visualization module for single nucleotide MVs and a convolutional neural network-based classification module for control-independent MV detection. DeepMosaic was trained on 180,000 simulated or experimentally assessed MVs, and was benchmarked on 619,740 simulated MVs and 530 independent biologically tested MVs from 16 genomes and 181 exomes. DeepMosaic achieved higher accuracy compared with existing methods on biological data, with a sensitivity of 0.78, specificity of 0.83 and positive predictive value of 0.96 on noncancer whole-genome sequencing data, as well as doubling the validation rate over previous best-practice methods on noncancer whole-exome sequencing data (0.43 versus 0.18). DeepMosaic represents an accurate MV classifier for noncancer samples that can be implemented as an alternative or complement to existing methods.

Testing for Phylogenetic Signal in Single-Cell RNA-Seq Data.

Phylogenetic methods are emerging as a useful tool to understand cancer evolutionary dynamics, including tumor structure, heterogeneity, and progression. Most currently used approaches utilize either bulk whole genome sequencing or single-cell DNA sequencing and are based on calling copy number alterations and single nucleotide variants (SNVs). Single-cell RNA sequencing (scRNA-seq) is commonly applied to explore differential gene expression of cancer cells throughout tumor progression. The method exacerbates the single-cell sequencing problem of low yield per cell with uneven expression levels. This accounts for low and uneven sequencing coverage and makes SNV detection and phylogenetic analysis challenging. In this article, we demonstrate for the first time that scRNA-seq data contain sufficient evolutionary signal and can also be utilized in phylogenetic analyses. We explore and compare results of such analyses based on both expression levels and SNVs called from scRNA-seq data. Both techniques are shown to be useful for reconstructing phylogenetic relationships between cells, reflecting the clonal composition of a tumor. Both standardized expression values and SNVs appear to be equally capable of reconstructing a similar pattern of phylogenetic relationship. This pattern is stable even when phylogenetic uncertainty is taken in account. Our results open up a new direction of somatic phylogenetics based on scRNA-seq data. Further research is required to refine and improve these approaches to capture the full picture of somatic evolutionary dynamics in cancer.

Simultaneous metabolite MALDI-MSI, whole exome and transcriptome analysis from formalin-fixed paraffin-embedded tissue sections

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows spatial analysis of proteins, metabolites, or small molecules from tissue sections. Here, we present the simultaneous generation and analysis of MALDI-MSI, whole-exome sequencing (WES), and RNA-sequencing data from the same formalin-fixed paraffin-embedded (FFPE) tissue sections. Genomic DNA and total RNA were extracted from (i) untreated, (ii) hematoxylin-eosin (HE) stained, and (iii) MALDI-MSI-analyzed FFPE tissue sections from three head and neck squamous cell carcinomas. MALDI-MSI data were generated by a time-of-flight analyzer prior to preprocessing and visualization. WES data were generated using a low-input protocol followed by detection of single-nucleotide variants (SNVs), tumor mutational burden, and mutational signatures. The transcriptome was determined using 3'-RNA sequencing and was examined for similarities and differences between processing stages. All data met the commonly accepted quality criteria. Besides SNVs commonly identified between differently processed tissues, FFPE-typical artifactual variants were detected. Tumor mutational burden was in the same range for tissues from the same patient and mutational signatures were highly overlapping. Transcriptome profiles showed high levels of correlation. Our data demonstrate that simultaneous molecular profiling of MALDI-MSI-processed FFPE tissue sections at the transcriptome and exome levels is feasible and reliable.The authors present a workflow that allows the simultaneous measurement of the whole exome and the transcriptome by next-generation sequencing from formalin-fixed paraffin-embedded tissue sections that were analyzed by matrix-assisted laser desorption ionization mass spectrometry imaging. The data and analyses demonstrate the feasibility and reproducibility of this approach, which expands the possibilities of multi-omics integration in cancer research.

Validation and Clinical Application of ONCOaccuPanel for Targeted Next-Generation Sequencing of Solid Tumors.

Targeted next-generation sequencing (NGS) is widely used for simultaneously detecting clinically informative genetic alterations in a single assay. Its application in clinical settings requires the validation of NGS gene panels. In this study, we aimed to validate a targeted hybridization capture-based DNA panel (ONCOaccuPanel) using the Illumina MiSeq sequencing platform. The panel allows the simultaneous detection of single-nucleotide variants (SNVs), insertions, deletions, and copy number changes of 323 genes and fusions of 17 genes in solid tumors. We used 16 formalin-fixed paraffin-embedded (FFPE) tumor samples with previously known genetic mutations and one reference material (HD827) for validation. Moreover, we sequenced an additional 117 FFPE tumor samples to demonstrate the clinical utility of this panel. Validation revealed a 100% positive percentage agreement and positive predictive value for the detection of SNVs, insertions, deletions, copy number changes, fusion genes, and microsatellite instability-high types. We observed high levels of reproducibility and repeatability (R2 correlation coefficients=0.96-0.98). In the limit of detection assessment, we identified all clinically relevant genes with allele frequencies > 3%. Furthermore, the clinical application of ONCOaccuPanel using 117 FFPE samples demonstrated robust detection of oncogenic alterations. Oncogenic alterations and targetable genetic alterations were detected in 98.2% and 27.4% cases, respectively. ONCOaccuPanel demonstrated high analytical sensitivity, reproducibility, and repeatability and is feasible for the detection of clinically relevant mutations in clinical settings.

Genomic Landscaping of Post-Transplant Lymphoproliferative Disorders Using Circulating Tumor DNA

Introduction: Identification of post-transplant lymphoproliferative disorder (PTLD) is a diagnostic challenge. The current diagnostic framework usually require analysis on tissue and imaging through PET/CT scans. Yet, these methods have diagnostic limitations. For instance due to missing information on tumor heterogeneity and relatively low sensitivity of PET/CT scans. Measuring cell free DNA (cfDNA) represents a novel, minimally-invasive method for tumor detection and genotyping. In this study we explored the feasibility of plasma cfDNA tumor genotyping with targeted deep sequencing to detect copy number variation (CNV), single nucleotide variants (SNVs) and Epstein Bar Virus (EBV) sequences, using a new in-house variant calling pipeline. Methods: In this multi-center observational cohort study of 18 patients diagnosed with monomorphic PTLD, we performed deep targeted NGS for SNV detection and low-coverage whole genome sequencing (lcWGS) for CNV detection. NGS was targeted on a panel of genes (n = 39) known to be recurrently mutated in B-cell lymphomas (244 kbp). All patients gave written informed consent. The R package CNAclinic was used to identify genome wide CNAs and fraction of the genome altered (FGA). The ichorCNA package was used to determine the ctDNA fraction in cfDNA samples. SNVs were called using a pipeline, combining 4 (cfDNA) SNV calling tools: Mutect2, LoFreq, VarDict & SiNVICT. Variants were filtered on presumed functional effect (Synonymous-, UTR-, Intron-, 2 kb upstream-, 2 kb downstream variants were removed). A "panel of normals” (variant blacklist) was generated from variants found in cfDNA from healthy controls, which was used to produce a variant blacklist to filter out technical artifacts and SNPs. Finally, variants with a variant allele frequency (VAF) < 1% and variants with an allele frequency (AF) of > 1% in 1000Genomes and/or gnomAD were filtered out. Results: The cohort included 18 PTLD patients. The majority of patients had stage IV disease (n=13, 76%). All of the PTLD cases were histologically proven, of diffuse large B cell lymphoma type and 10/18 patients (55%) were considered to have an EBV-negative tumor. EBV copy number in plasma was performed by qPCR in 12 patients and was considered to be elevated in four patients (>5000 copies/mL). The fraction of reads mapping to the EBV genome was compared between controls (n=8), EBV-negative PTLD (n=10) and EBV-positive PTLD (n=8). The median fraction of EBV reads for controls and EBV-negative PTLD were similar with a fraction of 1.5x10-4 (Q1-Q3: 1.3x10-4-1.6x10-4) and 1.8x10-4 (Q1-Q3: 1.4x10-4-3.9x10-4), respectively (p>0.05, Kruskal-Wallis). In EBV-positive tumors, the median fraction of EBV reads was 3-log higher, 5.3x10-1 (Q1-Q3: 7.3 x10-2-2) (p≤0.05, Kruskal-Wallis) CNVs were detected in cfDNA of 11/18 (61%) patients. Two of the patients in whom no CNVs were detected had initiated therapy at the time of plasma sampling. Median fraction of genome altered was higher in EBV-negative tumors (0.03, Q1-Q3: 2.5x10-4-0.12) than in EBV-positive tumors (5x10-4, Q1-Q3: 0-0.03), but this difference was not significant (p>0.05, Mann-Whitney U) (Figure 1). In the top 15 recurrently mutated genes (described below), across all 18 samples, 180 SNVs were found in total. The median amount of SNVs found in each sample was 11 (range 6-25). The most frequently mutated gene was CREPPB (12/18, 67%), in which the most prevalent mutation was p.K1497T (8/18, 44%). ARID1A was mutated in 10/18 cases (56%), followed by KMT2D (9/18, 50%). TET2, TP53, MEF2B & TNFAIP3 were all mutated in 7/18 cases (39%). SPEN & IRF2BP2 were mutated in 6/18 cases (33%). PIM1, NOTCH1, ATM, CARD11, DDX3X, IGLL5 were all mutated in 5/18 cases (28%) (Figure 2). Few of the mutations that are called in the top 15 recurrently mutated genes (8/180, 0.04%) lay close to 50% VAF, which might imply that these are SNPs not filtered out by the pipeline. Our SNV findings are comparable with previous literature, in which KMT2D, TET2 & TP53 are also amongst top-mutated genes (Menter et al., 2017), highlighting disruption of epigenetic regulation, NFkB pathway, and DNA damage response as drivers of PTLD. Conclusion: lcWGS in combination deep targeted sequencing allows for detection of CNV an SNV in diagnostic samples of patients with monomorphic PTLD. These data can potentially be used at screening, diagnosis and response monitoring, which is part of the ongoing prospective study NTR 7402. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

94. Comparison of FISH to whole exome/whole transcriptome detection of relevant structural alterations in Multiple Myeloma

In multiple myeloma and monoclonal gammopathies, single nucleotide variants (SNV), copy number variants (CNV) and IGH-rearrangements with prognostic/therapeutic relevance, are assessed by NGS, FISH, and karyotype. Recently, whole exome/transcriptome sequencing (WES/WTS) post CD138+-enrichment has been clinically implemented, thus we evaluated this approach to detect relevant alterations. Over 9 months, 160 aspirates were enriched including assessment of %CD138+ by flow cytometry post-enrichment. Extracted DNA/RNA were sent out for WES/WTS (20% tumor cellularity cut-off). Eighteen were insufficient where DNA yield correlated with WES/WTS success by Welch Two Sample t-test (p<0.001). Of 142 reported, post-enrichment %CD138 correlated with the detection of relevant SNV or CNV (n=97) (p<0.001), and after excluding those only displaying CHIP-associated SNVs (n=11) (p<0.00001). FISH was performed outside on similarly enriched CD138+ cells (collected during same procedure) for 122 cases. While the specificity was ?91%, the sensitivities of detection by WES of gain of 1q, 9, 11, 15 and loss of 1p, 13/13q14, 17p/17, were 70%, 79%, 76%, 79%, and 71%, 67%, 83% respectively. Lack of detection was primarily in cases with FISH-detected gain/loss <20%. For 10 cases with IGH-FGFR3 and 6 with IGH-MAF (by FISH), 9 had detectable IGH-NSD2 and 3 IGH-WWOX fusion transcripts respectively. Thus, WES/WTS is less sensitive than FISH for detection of CNV <20%, but affords the detection of additional relevant abnormalities (4p, 16q loss). Increasing the dataset will afford the establishment of cut-offs for DNA yield and %CD138+ to improve overall diagnostic yield, and for the level of CCND1 transcripts that correlate with IGH-CCND1 rearrangement.

Multimodal Urine Cell-Free DNA-Based Residual Disease Detection and Prognostication in Muscle-Invasive Bladder Cancer Patients

<h3>Purpose/Objective(s)</h3> Our group recently showcased a liquid biopsy assay (uCAPP-Seq) based on the detection of single nucleotide variants (SNVs) in urine cell-free DNA (cfDNA) from patients with muscle-invasive bladder cancer (MIBC). We found that assay positivity corresponded with the presence of residual cancer and a higher risk of disease recurrence despite curative-intent therapy (Chauhan et al., PLOS Medicine, 2021). Here, we explored multimodal urine-based genomics by integrating ultra-low-pass whole genome sequencing (ULP-WGS) and uCAPP-Seq for improving the sensitivity of residual disease detection and survival prediction among MIBC patients. <h3>Materials/Methods</h3> Urine samples from 74 MIBC patients were collected preoperatively on the day of radical cystectomy to assess urine tumor DNA (utDNA). We performed ULP-WGS of urine cfDNA from all 74 patients as well as 15 healthy adults. Tumor fraction (TF) based on genome-wide copy number alterations in urine cfDNA was estimated using ichorCNA. Variant allele frequency (VAF) based on SNVs was estimated by uCAPP-Seq. Pathologic complete response (pCR) was determined by surgical pathology. Multivariate logistic regression and random forest models were utilized to predict disease status based on VAF and TF levels, along with clinical variables (smoking, age, sex, race, and histology). Kaplan-Meier (KM) analysis was used to assess overall survival (OS) and progression-free survival (PFS). <h3>Results</h3> Mean utDNA VAF and TF levels were both significantly higher in patients without pCR compared to those who achieved pCR (11.9% vs. 4.0% VAF, p=0.044; 12.7% vs. 5.2% TF, p = 0.019) (Table 1). Our multimodal logistic regression model identified utDNA VAF (p < 0.001) and smoking status (p = 0.034) as significant predictors of disease status. Random forest modeling demonstrated that utDNA VAF and TF were both significant predictors. It achieved a sensitivity of 83% and a specificity of 79% for detecting residual disease. Strikingly, among the 15 patients with negative findings by both ULP-WGS and uCAPP-Seq, none experienced cancer progression (median follow-up time = 7.2 months). In contrast, the 59 patients with positive utDNA by ULP-WGS or uCAPP-Seq had significantly worse PFS (median = 6.0 months; HR = 3.54, p = 0.027). Moreover, utDNA positivity trended towards predicting significantly worse OS (HR = 3.43, p = 0.15). <h3>Conclusion</h3> Integration of ULP-WGS with uCAPP-Seq enabled robust detection of residual urine tumor DNA with high sensitivity and predicted progression-free survival in muscle-invasive bladder cancer patients. In the future, this type of multimodal urine-based genomic analysis may lead to more precise risk stratification and nonoperative clinical decision-making for muscle-invasive bladder cancer patients.