Somatic Single Nucleotide Variants Research Articles

Analytical and clinical validation of a custom 15-gene next-generation sequencing panel for the evaluation of circulating tumor DNA mutations in patients with advanced non-small-cell lung cancer.

BackgroundThis is a pilot proof-of-concept study to evaluate the utility of a custom 15-gene circulating tumor DNA (ctDNA) panel as a potential companion molecular next-generation sequencing (NGS) assay for identifying somatic single nucleotide variants and indels in non-small-cell lung cancer (NSCLC) patients. The custom panel covers the hotspot mutations in EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET, KIT, PDGFRA, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 genes which serve as biomarkers for guiding treatment decisions in NSCLC patients.MethodThe custom 15-gene ctDNA NGS panel was designed using ArcherDX Assay Designer. A total of 20 ng or 50 ng input ctDNA was used to construct the libraries. The analytical performance was evaluated using reference standards at different allellic frequencies (0.1%, 1%, 5% and parental). The clinical performance was evaluated using plasma samples collected from 10 treatment naïve advanced stage III or IV NSCLC patients who were tested for tissue EGFR mutations. The bioinformatics analysis was performed using the proprietary Archer Analysis Software.ResultsFor the analytical validation, we achieved 100% sensitivity and specificity for the detection of known mutations in the reference standards. The limit of detection was 1% allelic frequency. Clinical validation showed that the clinical sensitivity and specificity of the assay for detecting EGFR mutation were 83.3% and 100% respectively. In addition, the NGS panel also detected other mutations of uncertain significance in 6 out of 10 patients.ConclusionThis preliminary analysis showed that the custom 15-gene ctDNA NGS panel demonstrated good analytical and clinical performances for the EGFR mutation. Further studies incorporating the validation of other candidate gene mutations are warranted.

The genomic landscape across 474 surgically accessible epileptogenic human brain lesions.

Understanding the exact molecular mechanisms involved in the etiology of epileptogenic pathologies with or without tumor activity is essential for improving treatment of drug-resistant focal epilepsy. Here, we characterize the landscape of somatic genetic variants in resected brain specimens from 474 individuals with drug-resistant focal epilepsy using deep whole-exome sequencing (>350×) and whole-genome genotyping. Across the exome, we observe a greater number of somatic single-nucleotide variants (SNV) in low-grade epilepsy-associated tumors (LEAT; 7.92 ± 5.65 SNV) than in brain tissue from malformations of cortical development (MCD; 6.11 ± 4 SNV) or hippocampal sclerosis (HS; 5.1 ± 3.04 SNV). Tumor tissues also had the largest number of likely pathogenic variant carrying cells. LEAT had the highest proportion of samples with one or more somatic copy number variants (CNV; 24.7%), followed by MCD (5.4%) and HS (4.1%). Recurring somatic whole chromosome duplications affecting Chromosome 7 (16.8%), chromosome 5 (10.9%), and chromosome 20 (9.9%) were observed among LEAT. For germline variant-associated MCD genes such as TSC2, DEPDC5, and PTEN, germline SNV were frequently identified within large loss of heterozygosity regions, supporting the recently proposed 'second hit' disease mechanism in these genes. We detect somatic variants in twelve established lesional epilepsy genes and demonstrate exome-wide statistical support for three of these in the etiology of LEAT (e.g., BRAF) and MCD (e.g., SLC35A2 and MTOR). We also identify novel significant associations for PTPN11 with LEAT and NRAS Q61 mutated protein with a complex MCD characterized by polymicrogyria and nodular heterotopia. The variants identified in NRAS are known from cancer studies to lead to hyperactivation of NRAS, which can be targeted pharmacologically. We identify large recurrent 1q21-q44 duplication including AKT3 in association with focal cortical dysplasia type 2a with hyaline astrocytic inclusions, another rare and possibly under-recognized brain lesion. The clinical genetic analyses showed that the numbers of somatic SNV across the exome and the fraction of affected cells were positively correlated with the age at seizure onset and surgery in individuals with LEAT. In summary, our comprehensive genetic screen sheds light on the genome-scale landscape of genetic variants in epileptic brain lesions, informs the design of gene panels for clinical diagnostic screening, and guides future directions for clinical implementation of epilepsy surgery genetics.

Single-cell genome sequencing of human neurons identifies somatic point mutation and indel enrichment in regulatory elements.

Accurate somatic mutation detection from single-cell DNA sequencing is challenging due to amplification-related artifacts. To reduce this artifact burden, an improved amplification technique, primary template-directed amplification (PTA), was recently introduced. We analyzed whole-genome sequencing data from 52 PTA-amplified single neurons using SCAN2, a new genotyper we developed to leverage mutation signatures and allele balance in identifying somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) in PTA data. Our analysis confirms an increase in nonclonal somatic mutation in single neurons with age, but revises the estimated rate of this accumulation to 16 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels increase by at least three per year per neuron and are enriched in functional regions of the genome such as enhancers and promoters. Our data suggest that indels in gene-regulatory elements have a considerable effect on genome integrity in human neurons.

P11.46.A Whole exome sequencing identifies novel SLIT2 mutations in primary CNS lymphoma

Abstract Background Whole exome sequencing (WES) data on primary CNS lymphoma (PCNSL) is scarce and correlation with clinical observations is needed to facilitate personalized care. Here we present WES data of six PCNSL and correlate genetic findings with clinical outcome. Material and Methods WES was performed on paired blood and tumor DNA samples from six PCNSL patients to a median coverage of 100x (range: 73-135x) and 194x (range: 127-232x), respectively. Selected, novel mutations were validated with Sanger sequencing. Epstein-Barr virus (EBV) status was assessed in tumor tissue. Progression free survival (PFS) and overall survival (OS) were analyzed in our cohort and in an extension cohort from the cBio Cancer Genomics Portal using Kaplan-Meier method and log-rank tests. P values < 0.05 were considered significant. Results We found a median of 22 (range: 11-68) somatic copy number variations, 171 (range: 82-298) somatic single-nucleotide variants and 11 (range: 0-22) somatic insertions/deletions per PCNSL. CDKN2A loss, identified in five samples, was the most commonly detected copy number alteration. PCNSL carried mutations in previously described PIM1, MYD88, CD79B or BTG genes. In three PCNSL, we identified novel SLIT2 mutations (p.N63S, p.T590M, p.T732S) clustered around the leucine-rich repeats (LRR) domains. WNT-reporter and NF-kB reporter luciferase assays suggested alterations were loss-of-function variants. SLIT2 mutations were associated with shorter PFS in our cohort and with shorter OS in an extension cohort of 1856 lymphoid malignancies from the cBio Cancer Genomics Portal. Conclusion PCNSL harbored previously unknown SLIT2 mutations clustered around domains that are critical for interaction with ROBO proteins. SLIT2 mutations may be predictive of less favorable outcome in PCNSL. Additional clinical and molecular evaluation of these mutations is warranted.

Low-cost and clinically applicable copy number profiling using repeat DNA

BackgroundSomatic copy number alterations (SCNAs) are an important class of genomic alteration in cancer. They are frequently observed in cancer samples, with studies showing that, on average, SCNAs affect 34% of a cancer cell’s genome. Furthermore, SCNAs have been shown to be major drivers of tumour development and have been associated with response to therapy and prognosis. Large-scale cancer genome studies suggest that tumours are driven by somatic copy number alterations (SCNAs) or single-nucleotide variants (SNVs). Despite the frequency of SCNAs and their clinical relevance, the use of genomics assays in the clinic is biased towards targeted gene panels, which identify SNVs but provide limited scope to detect SCNAs throughout the genome. There is a need for a comparably low-cost and simple method for high-resolution SCNA profiling.ResultsWe present conliga, a fully probabilistic method that infers SCNA profiles from a low-cost, simple, and clinically-relevant assay (FAST-SeqS). When applied to 11 high-purity oesophageal adenocarcinoma samples, we obtain good agreement (Spearman’s rank correlation coefficient, rs=0.94) between conliga’s inferred SCNA profiles using FAST-SeqS data (approximately £14 per sample) and those inferred by ASCAT using high-coverage WGS (gold-standard). We find that conliga outperforms CNVkit (rs=0.89), also applied to FAST-SeqS data, and is comparable to QDNAseq (rs=0.96) applied to low-coverage WGS, which is approximately four-fold more expensive, more laborious and less clinically-relevant. By performing an in silico dilution series experiment, we find that conliga is particularly suited to detecting SCNAs in low tumour purity samples. At two million reads per sample, conliga is able to detect SCNAs in all nine samples at 3% tumour purity and as low as 0.5% purity in one sample. Crucially, we show that conliga’s hidden state information can be used to decide when a sample is abnormal or normal, whereas CNVkit and QDNAseq cannot provide this critical information.ConclusionsWe show that conliga provides high-resolution SCNA profiles using a convenient, low-cost assay. We believe conliga makes FAST-SeqS a more clinically valuable assay as well as a useful research tool, enabling inexpensive and fast copy number profiling of pre-malignant and cancer samples.

Landscape of somatic mutations in aging human heart muscle cells.

Using single-cell whole-genome sequencing, we identified and characterized the landscape of somatic single-nucleotide variants (sSNVs) in single cardiomyocytes from individuals across the human lifespan. Aged cardiomyocytes were found to have a higher burden of sSNVs and show mutational signatures that suggest failed repair of oxidative DNA damage.

Somatic single nucleotide variations and copy number variation can be used to distinguish high grade serous ovarian cancer from benign fallopian tubes with high accuracy (219)

Somatic single nucleotide variations and copy number variation can be used to distinguish high grade serous ovarian cancer from benign fallopian tubes with high accuracy (219)

Somatic mutations in single human cardiomyocytes reveal age-associated DNA damage and widespread oxidative genotoxicity

The accumulation of somatic DNA mutations over time is a hallmark of aging in many dividing and nondividing cells but has not been studied in postmitotic human cardiomyocytes. Using single-cell whole-genome sequencing, we identified and characterized the landscape of somatic single-nucleotide variants (sSNVs) in 56 single cardiomyocytes from 12 individuals (aged from 0.4 to 82 years). Cardiomyocyte sSNVs accumulate with age at rates that are faster than in many dividing cell types and nondividing neurons. Cardiomyocyte sSNVs show distinctive mutational signatures that implicate failed nucleotide excision repair and base excision repair of oxidative DNA damage, and defective mismatch repair. Since age-accumulated sSNVs create many damaging mutations that disrupt gene functions, polyploidization in cardiomyocytes may provide a mechanism of genetic compensation to minimize the complete knockout of essential genes during aging. Age-related accumulation of cardiac mutations provides a paradigm to understand the influence of aging on cardiac dysfunction.

SECEDO: SNV-based subclone detection using ultra-low coverage single-cell DNA sequencing.

MotivationSeveral recently developed single-cell DNA sequencing technologies enable whole-genome sequencing of thousands of cells. However, the ultra-low coverage of the sequenced data (<0.05× per cell) mostly limits their usage to the identification of copy number alterations in multi-megabase segments. Many tumors are not copy number-driven, and thus single-nucleotide variant (SNV)-based subclone detection may contribute to a more comprehensive view on intra-tumor heterogeneity. Due to the low coverage of the data, the identification of SNVs is only possible when superimposing the sequenced genomes of hundreds of genetically similar cells. Thus, we have developed a new approach to efficiently cluster tumor cells based on a Bayesian filtering approach of relevant loci and exploiting read overlap and phasing.ResultsWe developed Single Cell Data Tumor Clusterer (SECEDO, lat. ‘to separate’), a new method to cluster tumor cells based solely on SNVs, inferred on ultra-low coverage single-cell DNA sequencing data. We applied SECEDO to a synthetic dataset simulating 7250 cells and eight tumor subclones from a single patient and were able to accurately reconstruct the clonal composition, detecting 92.11% of the somatic SNVs, with the smallest clusters representing only 6.9% of the total population. When applied to five real single-cell sequencing datasets from a breast cancer patient, each consisting of 2000 cells, SECEDO was able to recover the major clonal composition in each dataset at the original coverage of 0.03×, achieving an Adjusted Rand Index (ARI) score of 0.6. The current state-of-the-art SNV-based clustering method achieved an ARI score of 0, even after merging cells to create higher coverage data (factor 10 increase), and was only able to match SECEDOs performance when pooling data from all five datasets, in addition to artificially increasing the sequencing coverage by a factor of 7. Variant calling on the resulting clusters recovered more than twice as many SNVs as would have been detected if calling on all cells together. Further, the allelic ratio of the called SNVs on each subcluster was more than double relative to the allelic ratio of the SNVs called without clustering, thus demonstrating that calling variants on subclones, in addition to both increasing sensitivity of SNV detection and attaching SNVs to subclones, significantly increases the confidence of the called variants.Availability and implementationSECEDO is implemented in C++ and is publicly available at https://github.com/ratschlab/secedo. Instructions to download the data and the evaluation code to reproduce the findings in this paper are available at: https://github.com/ratschlab/secedo-evaluation. The code and data of the submitted version are archived at: https://doi.org/10.5281/zenodo.6516955.Supplementary information Supplementary data are available at Bioinformatics online.

Targeted Next-Generation Sequencing of Thymic Epithelial Tumours Revealed Pathogenic Variants in KIT, ERBB2, KRAS, and TP53 in 30% of Thymic Carcinomas.

Simple SummaryThe biology of thymic epithelial tumours (TETs), including thymomas and thymic carcinomas, and particularly the extent of molecular dysregulation, is poorly understood. Through next-generation sequencing of 15 genes implicated in common solid tumours in 53 TETs, we found a larger number of single nucleotide variants (SNVs) in thymic carcinomas than thymomas. About 30% of thymic carcinomas had at least one somatic pathogenic gene variant in TP53, ERBB2, KIT, or KRAS, whereas variants of uncertain clinical significance in KIT, ERBB2, and FOXL2 were found exclusively in thymomas. The presence of somatic pathogenic variants was non-significantly associated with shorter disease-free survival in thymic carcinoma patients. No somatic pathogenic or likely pathogenic SNV was found in thymomas. Importantly, we also evaluated germline SNVs, adding to the number of known genetic alterations in TETs and thereby enhancing our molecular understanding of these neoplasms.A better understanding of the molecular pathogenesis of thymic epithelial tumours (TETs) could revolutionise their treatment. We evaluated thymomas and thymic carcinomas by next-generation sequencing (NGS) of somatic or germline single nucleotide variants (SNVs) in genes commonly mutated in solid tumours. In total, 19 thymomas and 34 thymic carcinomas were analysed for nonsynonymous SNVs in 15 genes by targeted NGS (reference genome: hg19/GRCh37). Ten SNVs in TP53 (G154V, R158P, L194H, R267fs, R273C, R306 *, Q317 *), ERBB2 (V773M), KIT (L576P), and KRAS (Q61L) considered somatic and pathogenic/likely pathogenic were detected in 10 of 34 (29.4%) thymic carcinomas. No somatic SNVs confirmed as pathogenic/likely pathogenic were found in thymomas. Rare SNVs of uncertain or unknown functional and clinical significance, to our knowledge not reported previously in TETs, were found in ERBB2 (S703R), KIT (I690V), and FOXL2 (P157S) in 3 of 19 (16%) thymomas. The most frequent germline SNVs were TP53 P72R (94% TETs), ERBB2 I655V (40% TETs), and KIT M541L (9% TETs). No significant difference in median disease-free survival (DFS) was found between thymic carcinoma patients with and without pathogenic SNVs (p = 0.190); however, a trend toward a longer DFS was observed in the latter (16.0 vs. 30.0 months, respectively). In summary, NGS analysis of TETs revealed several SNVs in genes related to the p53, AKT, MAPK, and K-Ras signalling pathways. Thymic carcinomas showed greater genetic dysregulation than thymomas. The germline and rare SNVs of uncertain clinical significance reported in this study add to the number of known genetic alterations in TETs, thus extending our molecular understanding of these neoplasms. Druggable KIT alterations in thymic carcinomas have potential as therapeutic targets.

RNA-SSNV: A Reliable Somatic Single Nucleotide Variant Identification Framework for Bulk RNA-Seq Data.

The usage of expressed somatic mutations may have a unique advantage in identifying active cancer driver mutations. However, accurately calling mutations from RNA-seq data is difficult due to confounding factors such as RNA-editing, reverse transcription, and gap alignment. In the present study, we proposed a framework (named RNA-SSNV, https://github.com/pmglab/RNA-SSNV) to call somatic single nucleotide variants (SSNV) from tumor bulk RNA-seq data. Based on a comprehensive multi-filtering strategy and a machine-learning classification model trained with comprehensively curated features, RNA-SSNV achieved the best precision–recall rate (0.880–0.884) in a testing dataset and robustly retained 0.94 AUC for the precision–recall curve in three validation adult-based TCGA (The Cancer Genome Atlas) datasets. We further showed that the somatic mutations called by RNA-SSNV tended to have a higher functional impact and therapeutic power in known driver genes. Furthermore, VAF (variant allele fraction) analysis revealed that subclonal harboring expressed mutations had evolutional selection advantage and RNA had higher detection power to rescue DNA-omitted mutations. In sum, RNA-SSNV will be a useful approach to accurately call expressed somatic mutations for a more insightful analysis of cancer drive genes and carcinogenic mechanisms.

Somatic Mutation of FAT Family Genes Implicated Superior Prognosis in Patients With Stomach Adenocarcinoma.

FAT family genes encode protocadherin, which regulates tumor cell proliferation and migration. Although transcriptional levels of FAT family members had been reported in multiple malignant tumors, the association between mutation and prognosis of the FAT family in stomach adenocarcinoma (STAD) has not been investigated. Herein, we performed a multi-omics integrative bioinformatics analysis using genomic and mRNA expression data to explore the role of gene mutations across the FAT family on clinical outcomes of STAD. The results showed that FAT mutations occurred in 174 of 435 (40%) of the samples. Patients with FAT mutations possessed significantly better progression-free survival (P = 0.019) and overall survival (P = 0.034) than those with non-FAT mutations, and FAT mutations exhibited significantly higher tumor mutational burden (TMB) and microsatellite instability. Notably, FAT mutations had a greater effect on somatic single-nucleotide variation than copy number variation and resulted in more abundant DNA damage repair (DDR) mutations. Further investigation demonstrated that FAT mutations contributed to an inflammatory tumor microenvironment (TME), as indicated by significantly increased numbers of activated CD4 and CD8 T cells, and significantly decreased numbers of mast cell, plasmacytoid dendritic cell, type 2 T helper cell, and high expression of immune-promoting genes. Moreover, biological process antigen processing and presentation, DNA replication, and DDR-related pathways were significantly upregulated in patients with FAT mutations. Collectively, FAT mutations significantly improved the survival of patients with STAD by enhancing tumor immunogenicity (e.g., TMB and DDR mutations) and an inflamed TME, indicating that the FAT family might be a potential prognostic and therapeutic biomarker for STAD.

P312: EMPLOYING WHOLE GENOME OPTICAL MAPPING TO CHARACTERIZE THE LANDSCAPE OF STRUCTURAL VARIANTS IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common subtypes are ETV6::RUNX1+ BCP-ALL, which harbor a reciprocal translocation between chromosome 12 and 21 leading to a juxtaposition of ETV6 to RUNX1, and high hyperdiploid (HHD) BCP-ALL harboring 51-67 chromosomes. In large-scale genomic studies, the mutational landscape of somatic single nucleotide variants and indels as well as copy number gains and losses based on array CGH have been well described in both subtypes. However, a comprehensive analysis of somatic structural variants (SVs) is still lacking which may reveal significant new insights on tumor development, progression, and treatment options in BCP-ALL. Aims: We aimed to primarily employ the novel technology whole genome optical mapping (WGOM) to unmask the landscape of structural variants in BCP-ALL. Firstly, we determined the concordance of chromosomal abnormalities identified by WGOM in comparison to standard techniques. Superiorly, we explored the landscape of somatic aberrations in ETV6::RUNX1+ and HHD tumors to identify recurrent alterations and novel SVs affecting genes not yet related to BCP-ALL pathogenesis. Methods: We extracted ultra-high molecular weight (UHMW) DNA of 13 ETV6::RUNX1+ and 18 HHD BCP-ALL with a tumor cell content of 25-96% at diagnosis. Additionally, we isolated DNA of fibroblasts or remission material of each pediatric patient as matching non-tumor control. Informed consent was obtained from all participants. DNA was fluorescently labeled by a sequence specific enzyme and subsequently linearized and imaged using a Saphyr instrument (Bionano Genomics). Using this approach, we generated comprehensive datasets of 31 matched tumor/non-tumor pairs, allowing the identification of somatically acquired SVs >500bp. In addition, corresponding karyotype, fluorescence in situ hybridization and SNP-array data of the leukemia samples were integrated and compared to WGOM data. Results: We validated 95% of 233 events observed by standard techniques with WGOM. All hallmark alterations including ETV6::RUNX1 fusion and high hyperdiploidy were identified by WGOM. In addition, we detected a median of 11 (1-36) somatically acquired events in leukemic cells with ETV6::RUNX1 fusion, compared to 3 (1-10) events in HHD cases. WGOM identified recurrent somatic structural aberrations occurring in at least three BCP-ALL cases, including focal deletions affecting ETV6 (9/31), PAX5 (5/31), ARPP21 (3/31), CD200/BTLA (3/31) and RAG2 (3/31). We detected subtype specific recurrent focal deletions disrupting ATF7IP (7/13) and BTG1 (5/13) in ETV6::RUNX1+ subclones whereas deletion of the CDKN2A/B locus (3/18) was only observed in HHD BCP-ALL. Interestingly, we discovered novel recurrently deleted regions on 12q24.11 (GPN3, 4/31) and Xq25 (STAG2, 3/31) in all BCP-ALL cases as well as focal loss of 19q13.11 (UBA2, 3/13) specific for ETV6::RUNX1+ subclones using WGOM. Moreover, complex three-way translocations in ETV6::RUNX1+ tumors were discovered by WGOM, including t(6;8;12)(p12.3;q24.23;q24.21) that leads to the disruption of MED13L and HMGCLL1. Summary/Conclusion: Our study showed that WGOM detects SVs as translocations and aneuploidies in BCP-ALL as good as conventional methods with a 95% concordance. However, WGOM allows a higher resolution and more precise localization of chromosomal breakpoints compared to standard techniques. Hence, we identified novel recurrent somatically acquired SVs including a deletion on Xq25 affecting STAG2, which might play an important role in pediatric BCP-ALL leukemogenesis.

Abstract 5118: Postoperative minimal residual disease to predict the risk of hepatocellular carcinoma (HCC) recurrence using plasma-only circulating tumor DNA assay

Abstract Background: Postoperative minimal residual diseases (MRD) are one of the main causes of postoperative short-term recurrence, which were difficult to be detected by conventional imaging examinations. Circulating tumor DNA (ctDNA) provides a novel approach for detecting the existence of MRD and predicting clinical outcomes of hepatocellular carcinoma (HCC). Most ctDNA MRD assays require tumor sequencing to identify tumor-derived mutations to facilitate ctDNA detection, requiring tumor and blood. Herein, we evaluated a plasma-only ctDNA assay integrating genomic signatures to enable tumor-uninformed MRD detection. Methods: 20 patients with hepatocellular carcinoma who underwent radical surgical resection were involved from July 2019 to October 2020. Blood samples were collected for next generation sequencing (NGS) within 7 days after surgery, and 10 of them had preoperatively matched plasma ctDNA results. NGS (381 genes panel) was performed on plasma samples to capture tumor somatic single-nucleotide variants (SNVs) and copy number variants (CNVs). Clinical information was obtained to evaluate the prognostic performance of ctDNA comparing with traditional clinical biomarkers. Plasma and peripheral blood cell samples were sequenced to a mean depth of 7396 and ctDNA results correlated with recurrence-free survival (RFS). Results: Characteristics of the 20 HCC patients were as follows: 85% age≥50 years, 70% HBV positive, 75% largest tumor diameter≥5cm, 55% liver cirrhosis, 85% AFP &amp;gt; 400ng/ml, 60% MVI, 55% PVTT, 50% BCLC C, 70% CNLC III. After a median follow-up of 13.60 months, 10 recurrence events were recorded. In order to evaluate the presence of MRD after radical surgery, at least one low frequency SNV detected was considered ctDNA-positive. Using stringent quality criteria 8/20 (20%) were ctDNA positive after resection. The median RFS for ctDNA-positive patients was 6.98 months versus 27.57 months for ctDNA-negative patients (HR 3.727, 95% CI 0.9461-14.65; p=0.0247). Combining postoperative ctDNA SNV and preoperatively ctDNA CNV features could increase the sensitivity for predicting prognosis. To further confirm the effect of MRD on the RFS of HCC patients, multiple Cox regression was used to assess the effect of multiple factors, including MRD, alpha-fetoprotein (AFP), HBV, Liver cirrhosis, macrovascular invasion, PVTT, Child-Pugh score and CNLC stage, on the RFS. The forest plot results showed that the postoperative ctDNA was an independent factor for predicting the RFS of patients with HCC (MRD: HR=14.02, p=0.018). Conclusion: These preliminary results suggest that ultra-deep sequencing of ctDNA analyses can detect somatic mutations to reflect postoperative MRD state and predict recurrence of HCC. Plasma-only MRD detection may be an effective and convenient approach for clinical prediction of HCC postoperative recurrence risk. Citation Format: Mingxin Pan, Jianpeng Cai, Yuyan Xu, Kaihang Zhong, Tingting Chen. Postoperative minimal residual disease to predict the risk of hepatocellular carcinoma (HCC) recurrence using plasma-only circulating tumor DNA assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5118.

Abstract 5119: Genomic landscape analysis in tumor tissue and circulating tumor DNA of Chinese patients with hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of tumor-associated death worldwide, owing to its high 5-year postoperative recurrence rate and heterogeneity. Although Molecular profiling has been used to predict prognosis and guide targeted therapy, obtaining liver tissue biopsies was still a clinical challenge of advanced HCC patients. Circulating tumor DNA (ctDNA) testing as a novel and convenient detecting techniques, which can revealed comprehensive information regarding to tumor genome profiles. The purpose of the study was to analyze and compare the genomic landscape characteristics of tumor tissue and ctDNA in Chinese HCC. Methods: 187 tumor tissue samples and 293 plasma samples from patients with HCC were collected from Nanfang hospital and 3DMed cohort, respectively. Next generation sequencing (NGS) assay (381/733 genes panel) was performed on tumor and plasma samples to capture tumor somatic single-nucleotide variants (SNVs). The gene variants information was summarized, analyzed and visualize using the maftools R package (Mayakonda et al. 2018.). Results: In total, DNA alterations were detected in 93.86% (275/293) plasma samples and 90.91% (170/187) in tumor samples, which variant levels included pathogenic, likely pathogenic and variant of uncertain significance. The most commonly altered genes in ctDNA results were TP53 (52%), TERT (24%), LRP1B (22%), FAT1 (15%), CTNNB1 (14%). While in HCC tumor testing results were TP53 (55%), TERT (27%), CTNNB1 (17%), SPTA1 (14%) and ARID1A (12%). Both the most variant classification was missense, next were frameshift indel. Both the top five affected pathways were TP53, RTK-RAS, NOTCH, WNT and Cell Cycle. In WNT pathway related genes, the highest alteration frequency tumor oncogene was CTNNB1, and suppressor gene was APC. We analyzed mutually exclusive and co-occurring event on top 25 mutated genes. In ctDNA analysis results, the probability of co-mutation between LRP1B and KMT2A gene was the highest (p &amp;lt; 0.001), while mutual exclusion between ARID1A and EGFR gene was the highest. Conclusion: Circulating tumor DNA mutation detection in HCC is feasible in the absence of HCC tissue DNA. The analysis results revealed the genomic landscape characteristics of blood-derived ctDNA in HCC were consistent with tumor tissues, which can provided HCC potential prognosis and treatment related genes information to further study. Citation Format: Dinghua Yang, Sheng Yu, Baitang Guo, Tingting Chen. Genomic landscape analysis in tumor tissue and circulating tumor DNA of Chinese patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5119.

Abstract 980: Genomic characterization of lymph node metastases in papillary thyroid carcinoma following the Chernobyl accident reveals an expression profile specific to metastatic process

Abstract Following the Chernobyl nuclear power plant explosion in Ukraine in 1986, increased childhood exposure to radioactive iodine (131I), which occurred primarily through contaminated food sources, has been consistently associated with increased risk of developing papillary thyroid carcinoma (PTC). Increased frequency of cervical lymph node metastases (LNM) is well recognized in pediatric PTC, including pediatric cases following the Chernobyl accident. We recently leveraged the collection of fresh-frozen tumor tissues from the Chernobyl Tissue Bank to conduct a genomic landscape analysis of 440 cases of PTC that provided new insights into radiation-related molecular characteristics of PTC occurring after the accident (Morton et al., Science 2021). Here, we extend that study to conduct a genomic landscape analysis of LNMs for a subset of 48 PTC cases to investigate the specific genomic alterations occurring in LNM. The analysis set comprised 144 samples, including fresh-frozen treatment-naïve primary and LNM PTC tumor samples as well as matched normal tissue or blood. Overall, the mutational load was highly concordant between primary and metastatic PTC. On average, 50% of somatic single nucleotide variants and 97% structural variants were shared between primary and metastatic PTCs. In contrast, the burden of somatic copy number alterations (SCNA) between primary tumor and LNM pairs was less concordant, particularly for copy number gains, whereas most of the copy number loss was found to be shared between matched pairs. PTC is usually driven by one driver mutation, most often involving the mitogen-activated protein kinase (MAPK) pathway. All driver mutations were shared between primary and metastatic PTC and were clonal; thus, no additional driver mutations were detected in metastatic PTC. Notably, however, the matched pairs in this study disproportionately had fusion drivers (77%), whereas only 23% of the drivers were BRAF V600E and none were RAS mutations. Transcriptome analysis revealed differentially expressed genes (DEGs) in metastatic PTC compared to primary PTC; three-quarters of the DEGs were overexpressed in metastatic PTC. Half of the LNM overexpressed DEGs were members of the HOXC family, which has been linked with epithelial-mesenchymal transition in cancer progression. There was also reduced expression in LNM for the DLX family, which relates to TGF-beta signaling. Our findings did not reveal a relationship between radiation dose and expression profiles in the LNM, comparable to our findings for the primary PTCs. We still observe that the efficiency of the radiation-induced PTC is paramount and subsequent events are directly related to the drivers in the MAPK pathway. For the cervical LNMs, we observed expression profiles not observed in the primary PTC that could give new insights into PTC local metastases. Citation Format: Olivia W. Lee, Danielle M. Karyadi, Chip Stewart, Tetiana I. Bogdanova, Jieqiong Dai, Stephen W. Hartley, Sara J. Schonfeld, Vidushi Kapoor, Marko Krznaric, Meredith Yeager, Amy Hutchinson, Belynda D. Hicks, Casey L. Dagnall, Julie M. Gastier-Foster, Jay Bowen, Mitchell J. Machiela, Elizabeth K. Cahoon, Kiyohiko Mabuchi, Vladimir Drozdovitch, Sergii Masiuk, Mykola Chepurny, Liudmyla Y. Zurnadzhy, Amy Berrington de González, Gad Getz, Gerry A. Thomas, Mykola D. Tronko, Lindsay M. Morton, Stephen J. Chanock. Genomic characterization of lymph node metastases in papillary thyroid carcinoma following the Chernobyl accident reveals an expression profile specific to metastatic process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 980.

Genomic Analysis Made It Possible to Identify Gene-Driver Alterations Covering the Time Window between Diagnosis of Neuroblastoma 4S and the Progression to Stage 4.

Neuroblastoma (NB) is a tumor of the developing sympathetic nervous system. Despite recent advances in understanding the complexity of NB, the mechanisms that determine its regression or progression are still largely unknown. Stage 4S NB is characterized by a favorable course of disease and often by spontaneous regression, while progression to true stage 4 is a very rare event. Here, we focused on genomic analysis of an NB case that progressed from stage 4S to stage 4 with a very poor outcome. Array-comparative genomic hybridization (a-CGH) on tumor-tissue DNA, and whole-exome sequencing (WES) on exosomes DNA derived from plasma collected at the onset and at the tumor progression, pointed out relevant genetic changes that can explain this clinical worsening. The combination of a-CGH and WES data allowed for the identification iof somatic copy number aberrations and single-nucleotide variants in genes known to be responsible for aggressive NB. KLRB1, MAPK3 and FANCA genes, which were lost at the time of progression, were studied for their possible role in this event by analyzing in silico the impact of their expression on the outcome of 786 NB patients.

Prognostic and predictive impact of circulating tumor DNA in advanced gastric cancer treated with immune checkpoint blockade.

e16019 Background: The utility of circulating tumor DNA (ctDNA) as a biomarker in patients with advanced gastric cancer (GC) receiving immune checkpoint blockade (ICB) is uncertain. Methods: Blood samples from advanced GC patients who received ICB were collected. The next-generation sequencing(NGS)–based targeted 733-gene and 61-gene panels (3D Medicines Inc.) were used to characterize somatic genomic alterations for baseline and on-treatment blood samples (4-12 weeks) respectively. For each sample, somatic single-nucleotide variants (SNV) and insertion/deletion mutations (indels) were used to calculate the mean variant allele frequency (VAF) and total mutation count. A VAF of 0.1% was the limit of detection, with the exception of variants detected in both pretreatment and on-treatment samples such that variants with VAF &lt; 0.1% in one sample were retained if the exact variant was also detected in the paired sample with VAF ≥0.1%. The variant genes at baseline were filtered against 61-genes panel list for calculating paired-baseline-VAF. The delta-VAF was calculated per patient as the sum of the on-treatment VAF minus the paired-baseline-VAF for each detected SNV or indels divided by the number of detected SNVs or indels. The percentage of molecular response was calculated per patient as delta-VAF divided by paired-baseline-VAF. Results: A total of 16 patients were was enrolled. The detection rate was 93.8% (15/16). The median total mutation count and baseline-VAF were 8 (range 0-44) and 3.13% (range 0-40.4%) respectively. The patients with top 50% baseline-VAF had shorter median PFS, compared to that with bottom 50% baseline-VAF (2.55 vs 4.85 months, p = 0.022, HR 2.98, 95%CI: 0.96-9.26). For baseline blood samples (N = 16), the median total mutation count and paired-baseline-VAF was 4 and 3.89% (range 0-40.4%) respectively. For on-treatment blood samples (N = 10), the median total mutation count and VAF were 3 (range 1-12) and 5.94% (0.30-37.12%) respectively. All the patients with progressive disease (PD, N = 6) have increased delta-VAF and all patients with stable disease (SD, N = 3) or partial response (PR, N = 1) have decreased delta-VAF. The median delta-VAF for PR, SD and PD were was -6.73%, -2.13% and 3.95% respectively. Compared with paired-baseline and on-treatment-VAF, the median percentage of molecular response for PR, SD and PD were -95.53%, -47.66% (range 7.08%-59.07%) and 138.73% (range 66.40%-347.18%). Compared with patients with increased VAF during treatment, the patients with decreased VAF have longer PFS (median 3.9 vs 1.75 months, p 0.004, HR, 0.24, 95%CI 0.06 to 0.97). Conclusions: This preliminary analysis contributes to understanding the role of ctDNA as a prognostic and predictive biomarker and its potential to complement radiologic endpoints. Further studies are warranted in larger cohort to confirm such observations for better management of GC.

Analytical validation of OriMIRACLE S, a personalized and tumor-informed approach to minimal residual disease surveillance.

e15013 Background: The presence of minimal residual disease (MRD) after curative-intent therapies in solid tumors is recognized as a risk factor for relapse. Glycoprotein-based tumor markers lack sensitivity and specificity for clinical application. Circulating tumor DNA (ctDNA) based MRD detection which can identify disease relapse ahead of radiological imaging has shown promising performance. The objective of this study was to develop and validate OriMIRACLE S (Minimal Residual Circulating Nucleic Acid Longitudinal Detection in Solid Tumor), a highly sensitive and specific tumor-informed assay for MRD detection via plasma cell-free DNA. Methods: Tumor-specific somatic single nucleotide variants (SNVs) were identified by whole exome sequencing of tumor tissue and matched germline DNA. Clonal SNVs were selected by OriSelector algorithm for patient-specific multiplex PCR-based NGS assay for MRD tracking. The limit of detection (LOD), specificity, sensitivity, repeatability, and reproducibility of this assay were determined experimentally with a series of standard references and in silico. Standard references were developed as following for assessing assay performance. The somatic variant references were two commercialized standards each containing five positive sites at variant allele frequency [VAF] of 0.1% and 1%, and a wild type sample. The single nucleotide polymorphism (SNP) references were made by blending three gDNA samples (160 sites) with gDNA samples without these SNPs to VAFs of 0%, 0.005%, 0.01%, 0.02%, 0.05% and 0.1%. And cfDNA references were made by titrating the clinical cfDNA mixture from five patients containing 31 positive sites with mononucleosomal HEK293 DNA to VAFs of 0.01-0.3% and 0.1-3%. Results: Site level sensitivity of ctDNA samples were 68.8% to 100% with 10 to 50ng input ctDNA (3,000 to 15,000 haploid genome equivalents). Site level reproducibility and repeatability were 98.0% and 97.0% using somatic variant references, respectively. The detection of three positive sites was sufficient to achieve a nearly 100% overall sample level sensitivity and specificity (6,000 haploid genome equivalents of gDNA samples) determined either by calculating a binomial probability or in silico with customized panels have 21 to 30 variants. The LOD of this assay was as low as 0.01% with the algorithm of OriCleverClover which could filter process and sequencing-related artifacts. Conclusions: OriMIRACLE S has been analytically validated as a highly sensitive and specific method to detect low quantities of ctDNA in plasma. The low limit of detection at a VAF of only 0.01% with low DNA input. OriMIRACLE S has wide-ranging potential applications in the clinical setting and drug development in a real-world scenario.

Longitudinal circulating tumor DNA (ctDNA) whole-exome sequencing (WES) in the phase Ib/II trial of palbociclib and bazedoxifene reveals genomic dynamics and clonal evolution with the acquisition of treatment resistance in hormone receptor-positive, HER2-negative (HR+ HER2-), advanced breast cancer (ABC).

1058 Background: Patients (pts) with HR+ HER2- ABC ultimately develop endocrine resistance. To gain insights into the genetic mechanisms of resistance we performed WES on serial plasma samples from endocrine resistant pts treated on a clinical trial (NCT02448771). Methods: Plasma samples were collected at baseline (n=36), day 1 of cycle 2 (n=33), and at the end of treatment (EOT, n=33). Samples were subjected to ultra-low passage (ULP, 0.19-0.57X) WGS to determine ctDNA tumor fraction (TF) for the selection of samples (TF&gt;0.03) for subsequent WES (193X). Somatic single nucleotide variations, somatic copy number alteration (SCNA), phylogeny, tumor mutational burden, mutational signatures, and germline analyses were performed. Results: All 102 samples underwent successful ULP and 68 WES. Overall, most frequent pathogenic mutations were in ESR1 and PIK3CA. At baseline, 32% of pts had ESR1 mutation and 21% PIK3CA mutation. There was no association between ESR1 mutations and PFS. In contrast, baseline PIK3CA mutations were detected only in pts who did not have a clinical benefit, and were associated with worse PFS compared to pts with wild-type PIK3CA (1.8 vs. 3.9 months, respectively, HR=0.2, 95% CI 0.06-0.6, P=0.0019, log-rank test). Additionally, pts with a baseline truncating mutation, mostly in tumor suppressor genes ( TP53, MEN1, RB1, CDKN1B, NF1, TP53BP1, TP63, SMAD2/4, ARID1A, KMT2C), also had a significantly worse PFS (1.7 vs 3.8 months, HR=0.3, 95% CI 0.1-0.7, P=0.006, log-rank test). At EOT, 20% (4/20) of pts with matched baseline samples had newly acquired mutations that are suggestive of mechanisms of acquired resistance and offer potential therapeutic targets (e.g. ERBB2, PIK3CA). SCNA analysis showed that in all pts there were at least 2 SCNAs in cancer-related driver genes, most common in CCND1 and ELF3. Moreover, in all samples we identified at least 1 SCNA related to a potential mechanism of resistance. To better understand tumor heterogeneity and sub-clonal architecture we performed an evolutionary analysis (sufficient TF≥0.15, available in n=7). Phylogenetic analysis revealed sub-clonal dynamics that could explain the acquisition of resistance in at least three pts (3/7), and identified novel genes which might have role in endocrine resistance (e.g. DCAF13, ZFHX3). Conclusions: Our results demonstrate the feasibility and utility of serial WES in a clinical trial. Serial ctDNA WES and evolutionary studies enabled us to discover novel potential genomic mechanisms of tumor progression, and identified PIK3CA mutations as a candidate biomarker of resistance to the combination of palbociclib and bazedoxifene, which may apply to other next generation endocrine treatments. Clinical trial information: NCT02448771.