Number Of Single Nucleotide Variants Research Articles

HiChIP-based Epigenomic Footprinting Identifies a Promoter Variant of UXS1 that Confers Genetic Susceptibility to Gastroesophageal Cancer.

Genome-wide association studies (GWAS) have identified more than a hundred single nucleotide variants (SNVs) associated with the risk of gastroesophageal cancer (GEC). The majority of the identified SNVs map to noncoding regions of the genome. Uncovering the causal SNVs and the genes they modulate could help improve GEC prevention and treatment. Here, we used HiChIP against histone 3 lysine 27 acetylation (H3K27ac) to simultaneously annotate active promoters and enhancers, identify the interactions between them, and detect nucleosome free regions (NFRs) harboring potential causal SNVs in a single assay. Application of H3K27Ac HiChIP in GEC relevant models identified 61 potential functional SNVs that reside in NFRs and interact with 49 genes at 17 loci. The approach led to a 67% reduction in the number of SNVs in linkage disequilibrium at these 17 loci, and at seven loci a single putative causal SNV was identified. One SNV, rs147518036, located within the promoter of the UDP-glucuronate decarboxylase 1 (UXS1) gene appeared to underlie the GEC risk association captured by the rs75460256 index SNV. The rs147518036 SNV creates a GABPA DNA recognition motif, resulting in increased promoter activity, and CRISPR-mediated inhibition of the UXS1 promoter reduced viability of GEC cells. These findings provide a framework that simplifies the identification of potentially functional regulatory SNVs and target genes underlying risk-associated loci. In addition, the study implicates increased expression of the enzyme UXS1 and activation of its metabolic pathway as a predisposition to gastric cancer, which highlights potential therapeutic avenues to treat this disease.

Abstract IA016: Molecular diversity of uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer with a high recurrence rate and poor prognosis disproportionately affecting black women. Whole-exome-sequencing analysis revealed that up to 10% of USC harbor features of ultra/hypermutation due to a deficiency of polymerase e (POLE) and/or mismatched-repair genes (MMRd). These tumors are notable for having a high number of single nucleotide variants (SNV), no LOH segments or copy-number variants (CNV) and a peculiar genetic signature characterized by an increased rate of C > A transversions. In contrast, the overwhelming majority of USC (~ 90%) are POLE/MMR proficient, have a low number of SNV and demonstrate alteration in genes including TP53, PIK3CA mutation/amplification, ERBB2 amplification, CCNE1 amplification, FBXW7 mutation/deletion, PPP2R1A mutation, and somatic mutations involving homologous recombination genes. Frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling-complex, and TAF1, an element of the core TFIID transcriptional machinery are also detected in a significant number of USC. Somatic CNVs, a sign of genomic instability, are identified in the majority of USC with multiple copy number gains and copy number losses, with the most frequent events taking place in pathways similar to those found from analysis of SNVs. In addition to loss of TP53, frequent deletion of a small segment of chromosome 19 containing MBD3, a member of the NuRD-chromatin-modification-complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (encoding for the HER2/neu receptor) and CCNE1 (a target of FBXW7-mediated ubiquitination) are commonly detected. Overall, these findings suggest that both somatic point mutations (SNV) and somatic CNV play a major role in the pathogenesis of USC. Importantly, the frequent mutation of DNA damage, chromatin remodeling, cell cycle and cell proliferation pathways in USC including the overexpression/amplification of HER2/neu, CCNE1 and the tumor-associated calcium signal transducer 2 (TACSTD2/TROP2) genes, suggest novel targets for therapy against this lethal variant of endometrial cancer. Citation Format: Alessandro D. Santin. Molecular diversity of uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA016.

Comparison of the DNBSEQ platform and Illumina HiSeq 2000 for bacterial genome assembly

The Illumina HiSeq platform has been a commonly used option for bacterial genome sequencing. Now the BGI DNA nanoball (DNB) nanoarrays platform may provide an alternative platform for sequencing of bacterial genomes. To explore the impact of sequencing platforms on bacterial genome assembly, quality assessment, sequence alignment, functional annotation, mutation detection, and metagenome mapping, we compared genome assemblies based on sequencing of cultured bacterial species using the HiSeq 2000 and BGISEQ-500 platforms. In addition, simulated reads were used to evaluate the impact of insert size on genome assembly. Genome assemblies based on BGISEQ-500 sequencing exhibited higher completeness and fewer N bases in high GC genomes, whereas HiSeq 2000 assemblies exhibited higher N50. The majority of assembly assessment parameters, sequences of 16S rRNA genes and genomes, numbers of single nucleotide variants (SNV), and mapping to metagenome data did not differ significantly between platforms. More insertions were detected in HiSeq 2000 genome assemblies, whereas more deletions were detected in BGISEQ-500 genome assemblies. Insert size had no significant impact on genome assembly. Taken together, our results suggest that DNBSEQ platforms would be a valid substitute for HiSeq 2000 for bacterial genome sequencing.

Transcriptomic and genomic profiling of multiple primary colorectal cancers reveals intratumor heterogeneity and a distinct immune microenvironment

This study reported on the intratumor genomic and immunological heterogeneity of different tumor lesions from a single patient with multiple primary colorectal cancer (MPCC). The goal of this study was to explore the molecular and microenvironment characteristics of tumor lesions from different primary sites in a patient with MPCC.A total of three tumor lesions located in the hepatic flexure of the transverse colon, sigmoid colon, and rectum were collected from a 72-year-old male patient with MPCC. All three tumor samples were examined by using whole-exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq). The transcriptome data of The Cancer Genome Atlas (TCGA) colon cancer (COAD) dataset were explored to characterize the biological impacts of certain immune cells. Only three nonsynonymous mutations were shared by all of the tumor lesions, whereas a number of single nucleotide variant (SNV) and copy number variation (CNV) mutations were shared by tumor samples from the sigmoid colon and rectum. Transcriptomic analysis showed that tumor lesions derived from the transverse colon had decreased levels of RTK, ERK, and AKT pathway activity, thus suggesting lower oncogenic properties in the transverse lesion compared to the other two samples. Further immune landscape evaluation by using single-cell transcriptomic analysis displayed significant intratumor heterogeneity in MPCC. Specifically, more abundant mucosal-associated invariant T (MAIT) cell infiltration was found in transverse colon tumor lesions. Afterwards, we found that higher MAIT cell infiltration may correlate with a better prognosis of patients with colon cancer (immunohistochemical status was MSI-L/pMMR) by using a publicly available TCGA dataset.

Impact of Genomic Alterations on the Clinical Outcome of Patients With Hepatitis B-related Hepatocellular Carcinoma Receiving Curative Surgery: A Retrospective Cohort Study.

The aim of the current study was to obtain comprehensive genomic information on viral hepatitis B (HBV)-related hepatocellular carcinoma (HCC) and identify potential biomarkers of early recurrence in patients receiving curative surgery. A total of 104 patients with HBV-related HCC receiving curative surgery at Kaohsiung Chang Gung Memorial Hospital between January 2017 and December 2020 were identified, including 52 patients each with and without recurrence. Next-generation sequencing was performed to investigate genomic alterations caused by surgical resection of specimens. The Kaplan-Meier method was used to estimate disease-free survival and overall survival. The landscape of gene mutations in HCC patients of our cohort showed a median number of single nucleotide variants of 250, a median number of insertions and deletions of 22, and a median number of protein-coding mutations of 185. The 10 most frequently mutated genes were TP53 (43%), TTN (39%), MUC16 (28%), PCLO (25%), OBSCN (22%), ADGRV1 (19%), ALB (18%), SYNE1 (18%), DNAH17 (17%), and RYR1 (17%). The tumour mutation burden was 4.8 mutations per megabase, and high microsatellite instability was reported in only three patients. In addition, the mutational signatures showed that aristolochic acid exposure was highly implicated in our HCC cohort. Five mutant genes, TBC1D4, ITGA4, RPS6KA3, VWA8, and FMN2, were more frequent in the recurrence group than that in the non-recurrence group. Our results present an in-depth genomic analysis of HBV-related HCC. The study findings provide an improved understanding of the related molecular mechanisms and identify potential biomarkers associated with early tumour recurrence after curative resection.

Abstract PO-075: Immunosuppression allows for proliferation of alternative splicing events in a murine model of tobacco-associated oral cavity squamous cell carcinoma

Abstract Background: Non-canonical alternative splicing events have been demonstrated to occur in head and neck squamous cell carcinoma, and result in functionally active aberrant isoforms that contribute to carcinogenesis. Our preliminary data also demonstrates that high splicing burden is associated with higher recurrence rates in oral cancer as well as relative immunosuppression. These splicing events are hypothesized to generate immunogenic neoantigens given non-self sequences that are generated from these novel isoforms. However, the role of the immune system in regulating the proliferation of aberrant splicing is not well understood. Therefore, we evaluated splicing alteration in the setting of immunosuppression in a murine model of oral cancer. Study Design: An established murine cell line with genetic signature resembling human tobacco-associated oral cavity SCC (4MOSC1) was injected into the oral tongue of wild type (C57/Blk6) and immunocompromised (SCID) mice. At takedown, specimens were collected for bulk RNA sequencing (RNASeq). Fastq files were aligned to mm39 using STAR v.2.7.1a. Splicing analysis was performed using OutSplice. Somatic variants in tumor samples were determined from RNASeq data using the GATK v4.4.0 tool Mutect2. Mutational burden was estimated using the number of single nucleotide variants (SNVs) called in each sample (CountVariants, GATK). Trinity v2.12.0 and NetMHCPan v4.1 were used to define PHBR scores to predict MHC binding affinity of neopeptides. Results: Seven of ten mice had measurable tumor by day 5, for an engraftment rate of 70% (3/5 C57/Blk6; 4/5 SCID). At day 15, tumors in wild type mice had a mean volume of 14.7mm3 compared to 28.9mm3 in SCID mice (one-sided p=0.132). Sequencing analysis demonstrated that splicing burden was higher in tumors developed in SCID mice compared to Blk6 mice (mean 475.25 vs 381.67, p=0.022). The mean number of SNVs were 144.2 mt/mb in SCID mice compared to 81.2 mt/mb in Blk6 mice (p=0.044). Amongst predicted splice derived neopeptides, those in SCID tumors had lower PHBR scores versus wild type tumors (median: 3.13 vs 4.23, p<0.001), correlating with higher predicted MHC binding affinity. However, amongst very strong binders (PHBR score ≤0.5), there was no difference in PHBR score between groups (0.30 vs 0.22, p=0.97). Conclusion: As expected, immunosuppression allowed for increased growth of tumors in the murine oral cancer model. We show that immunosuppression allows for the proliferation of both alternative splicing events and mutations. These preliminary data demonstrate that the immune system may play a role in regulating aberrant splicing events in oral cancer. Aberrant splice derived peptides which stimulate the immune system may be more likely to develop in an immunosuppressed setting. Citation Format: Mitchell Victor, Sophie Jang, Joseph Bendik, Silvio Gutkind, Theresa Guo. Immunosuppression allows for proliferation of alternative splicing events in a murine model of tobacco-associated oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-075.

Transcriptomic Analysis of Medicago truncatula under Long-Day Conditions

The objective of this research was to understand the expression characteristics and biological functions of Medicago truncatula genes under long-day conditions. The leaves of “R108” tribulus Medicago truncatula at the branch stage (A), bud stage (B), initial flowering stage (C), and full flowering stage (D) were sequenced using RNA-Seq technology. The genome of Medicago truncatula, a related species of Medicago truncatula, was used as the reference genome for sequence comparison. The transcriptomes of three adjacent periods (A vs. B, B vs. C, and C vs. D) were analyzed for differential gene expression and these genes were screened. A total of 6875 differentially expressed genes were detected. GO functional analysis showed that differentially expressed genes were mainly involved in biological processes, cell components, and molecular functions, among which the most differentially expressed genes were involved in the synthesis of cell components. KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in circadian rhythm, photosynthetic antenna protein, ribosome metabolism, and other pathways. The number of single nucleotide variants detected by cSNP analysis was 312,875, and the frequency of A/G and C/T were the highest. The function of eggNOG was divided into 23 categories, with a total of 26,745 genes having similarities, while 9008 genes were classified as having an unknown function, 2669 genes were classified as part of signal transduction mechanisms, and 2194 genes were classified as being involved in transcription. In different developmental stages (A vs. B, B vs. C, and C vs. D), 3463 up-regulated and 3412 down-regulated differentially expressed genes were found. The difference between up-regulated and down-regulated genes was more noteworthy at the bud stage and the initial flowering stage. In addition, a total of 79 flowering genes were found, of which 51 differential genes were identified as participating in the photoperiodic regulation pathway, consisting of 23 differential genes that were up-regulated, and 28 differential genes that were down-regulated. The ratios of gene-LOC11410562(GI), gene-LOC11435974(CO), gene-LOC11422615(TOC1), and gene-LOC11432385(LHY) were higher than those of gene-LOC25500742(PHYA) and gene-LOC11 431402(ELF3); gene-LOC11434778(Col13), gene-LOC25498015(Col6), and gene-LOC11415514(Col9) were pre-expressed. The above differentially expressed genes were significantly expressed in different developmental stages of Medicago truncatula, which lays a foundation for further study of the molecular mechanism of Medicago truncatula.

ZFP37, C3H, NAC94, and bHLH148 transcription factors regulate cultivar-specific drought response by modulating r40C1 gene expression in rice

Lectins are gradually gaining importance in regulating abiotic stress response in plants. Among them, the R40 family plays pivotal role in regulating drought response. The expression of the Osr40C1 positively correlates with the degree of drought tolerance in indica rice cultivars. Although the mechanism of Osr40C1-mediated drought tolerance is well characterized, the gene's regulation under drought remains obscure. Here, we demonstrate that the Osr40C1 promoter is activated under drought to induce its transcription. A number of single nucleotide variations in the Osr40C1 promoter of the drought-tolerant IR36 and sensitive MTU1010 cultivar are detected. However, promoter swapping analysis reveals that these variations in the promoter sequence do not explain the cultivar-specific expression pattern of this gene. Next, we identified the transcription factors that interact with the Osr40C1 promoter. Among these transcription factors, OsZFP37, OsC3H, OsNAC94, and OsbHLH148 display significantly higher transcript abundance in IR36 compared with MTU1010. Furthermore, overexpression of each of these transcription factors confers drought tolerance, whereas their silencing leads to drought sensitivity. Interestingly, the Osr40C1 expression is significantly up-regulated in the transcription factor overexpression lines, while its expression is undetectable in the silencing lines. Together our study highlights how different transcription factors regulate the cultivar-specific expression pattern of Osr40C1 in rice.

Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3+/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3+/− and Phlda3−/− littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Critically short telomeres derepress retrotransposons to promote genome instability in embryonic stem cells

Telomeres, at the ends of chromosomes, protect chromosomes from fusion and preserve genomic stability. However, the molecular mechanisms underlying telomere attrition-induced genome instability remain to be understood. We systematically analyzed the expression of retrotransposons and performed genomic sequencing of different cell and tissue types with telomeres of varying lengths due to telomerase deficiency. We found that critically short telomeres altered retrotransposon activity to promote genomic instability in mouse embryonic stem cells, as evidenced by elevated numbers of single nucleotide variants, indels and copy number variations (CNVs). Transpositions of retrotransposons such as LINE1 resulting from the short telomeres can also be found in these genomes with elevated number of mutations and CNVs. Retrotransposon activation is linked to increased chromatin accessibility, and reduced heterochromatin abundance correlates with short telomeres. Re-elongation of telomeres upon recovery of telomerase partly represses retrotransposons and heterochromatin accumulation. Together, our findings suggest a potential mechanism by which telomeres maintain genomic stability by suppressing chromatin accessibility and retrotransposon activity.

Multimarker omnibus tests by leveraging individual marker summary statistics from large biobanks.

As biobanks become increasingly popular, access to genotypic and phenotypic data continues to increase in the form of precomputed summary statistics (PCSS). Widespread accessibility of PCSS alleviates many issues related to biobank data, including that of data privacy and confidentiality, as well as high computational costs. However, questions remain about how to maximally leverage PCSS for downstream statistical analyses. Here we present a novel method for testing the association of an arbitrary number of single nucleotide variants (SNVs) on a linear combination of phenotypes after adjusting for covariates for common multimarker tests (e.g., SKAT, SKAT-O) without access to individual patient-level data (IPD). We validate exact formulas for each method, and demonstrate their accuracy through simulation studies and an application to fatty acid phenotypic data from the Framingham Heart Study.

Echoed induction of nucleotide variants and chromosomal structural variants in cancer cells

Generally, the number of single-nucleotide variants (SNVs) in somatic cells increases with age, which is expected for replication errors. The number of SNVs in cancer cells, however, is often much higher than that in somatic cells, raising the question of whether cancer cells possess SNV induction pathways. The present study shows that the number of SNVs in cancer cells correlates with the number of chromosomal structural variants (SVs). While Kataegis, localized hypermutations typically arising near SV sites, revealed multiple SNVs within 1 kb, SV-associated SNVs were generally observed within 0.1–1 Mb of SV sites, irrespective of Kataegis status. SNVs enriched within 1 Mb of SV regions were associated with deficiency of DNA damage repair, including HR deficiency-associated single base substitution 3 (SBS3) and exogenous damage-associated SBS7 and SBS36 signatures. We also observed a similar correlation between SVs and SNVs in cells that had undergone clonal evolution in association with genomic instability, implying an association between genomic instability and SV-associated induction of SNVs.

The ctDNA-based postoperative molecular residual disease status in different subtypes of early-stage breast cancer.

Breast cancer is a highly heterogeneous disease. Early-stage, non-metastatic breast cancer is considered curable after definitive treatment. Early detection of tumor recurrence and metastasis through sensitive biomarkers is helpful for guiding clinical decision-making and early intervention in second-line treatment, which could improve patient prognosis and survival. In this real-world study, we retrospectively analyzed 82 patients with stages I to III breast cancer who had been analyzed by molecular residual disease (MRD) assay. A total of 82 tumor tissues and 224 peripheral blood samples were collected and detected by next-generation sequencing (NGS) based on a 1,021-gene panel in this study. MRD positivity was detected in 18 of 82 patients (22.0%). The hormone receptor-/human epidermal growth factor receptor 2+ (HR-/HER2+) subgroup had the highest postoperative MRD detection rate at 30.8% (4/13). The BRCA2 and SLX4 genes were significantly enriched in all patients in the MRD positive group and FGFR1 amplification was significantly enriched in the MRD negative group with HR+/HER2-. The number of single nucleotide variants (SNVs) in tissue samples of MRD-positive patients was higher than that of MRD-negative patients (11.94 vs. 8.50 SNVs/sample). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that there was a similar biological function of the tumor-mutated genes in the 2 MRD status groups. This real-world study confirmed that patient samples of primary tumor tissue with different MRD status and molecular subtypes had differential genetic features, which may be used to predict patients at high risk for recurrence.

P12.10.A Supratentorial glioblastoma with spinal metastasis: a case report with molecular profiling

Abstract Background Glioblastoma (GBM) is regarded as an aggressive brain tumor that rarely develops extracranial metastases. Despite well-investigated molecular alterations in GBM, there is a limited understanding of these associated with the metastatic potential of GBM. Material and Methods We present a case report of a 43-year-old woman with frontal GBM IDH-wildtype, who underwent gross-total resection followed by chemoradiation. Five months after surgery, the patient was diagnosed with an intraspinal GBM metastasis. Next-generation sequencing analysis of both primary and metastatic GBM tissues was performed using the Illumina TruSight Tumor 170 assay. Results The number of single nucleotide variants observed in the metastatic sample was more than 2-times higher. Mutations in TP53, PTEN, and RB1 found in the primary and metastatic tissue samples are indicative of the mesenchymal GBM subtype. Among others, in the metastatic sample, there were detected two inactivating mutations (Arg1026Ile, Trp1831Ter) in the NF1 gene, two novel NOTCH3 variants of unknown significance predicted to be damaging (Pro1505Thr, Cys1099Tyr), one novel ARID1A variant of unknown significance (Arg1046Ser), and one gene fusion of unknown significance EIF2B5-KIF5B. Conclusion Based on the literature evidence, the inactivation of NF1, NOTCH3, and ARID1A could explain, at least in part, the acquired invasiveness and metastatic potential in this particular GBM case. This work was supported by the grant MUNI/A/1408/2021 of the Masaryk University, Brno, Czech Republic, and INTER-EXCELLENCE Programme / INTER-COST project no. LTC20027 of the Ministry of Health, Czech Republic.

Abstract 3992: Modular ddPCR assay design for highly sensitive single nucleotide variant detection

Abstract Introduction: Single-nucleotide variants (SNVs) are key drivers of both oncogenesis and emergent resistance to targeted therapies. Although high-throughput Identification of SNVs can be achieved with Next Generation Sequencing (NGS) technologies, the cost is significant, and proportional to the desired sensitivity of variant detection. Conversely, while only small numbers of SNVs can be queried in an individual quantitative PCR (qPCR) or droplet digital PCR (ddPCR) reaction, a fully optimized qPCR or ddPCR assay can detect SNVs at very low frequencies for a fraction of the cost of sequencing. Herein, we present a generalizable approach to PCR assay design, using a combination of mutation-sensitive induced mismatch primers, wild-type suppressors, and TaqMan probes, that permits detection of SNVs below 0.1%. Method: Known oncogenic hotspot regions were selected for development of mutation-detecting PCR assays. For each hotspot, a mutation-specific-mismatch primer, suppressor, and probe (MSMPSP) cassette was designed consisting of 1) a mutation-bearing forward primer with engineered mismatches, 2) a wild-type, extension-locked blocker oligo, 3) a fluorescent TaqMan probe, and 4) a downstream reverse primer. Designed mutation assays were evaluated using plasmid control sequences, remnant clinical specimens, and healthy donor peripheral blood samples spiked with known quantities of mutation-bearing plasmid or cell lines and processed on the BioRad AutoDG/QX200 droplet digital PCR system. Results: Variant detection below 0.1% was achieved for all tested mutations, including those residing in homopolymeric and GC-rich regions, from as low as 100 ng DNA input, in a single day. At higher variant frequencies accessible to multiple platforms, results with MSMPSP-based ddPCR assays exhibited excellent concordance with NGS and qPCR results and yielded linear signal decline down to 0.01% variant allele frequency. Overall assay sensitivity was proportional to DNA input, and improved detection limits were achieved by increased DNA input and combined well analysis. Conclusion: Utilizing the MSMPSP approach, coupled with ddPCR, variant detection for targeted mutations below 0.1% is achievable in diverse genomic contexts. Furthermore, simple design principles and flexible probe-binding sites permit rapid development of new assays, allowing critical mutations to be tracked as they are discovered. While multiplexing capabilities are reduced compared with NGS, short time-to-results, coupled with low cost-per-sample for MSMPSP ddPCR, permits faster treatment decisions at reduced cost to the healthcare system. Citation Format: Sarah Heine, Ashley Beams, Nathan Riccitelli, Shabnam Tangri. Modular ddPCR assay design for highly sensitive single nucleotide variant detection [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 3992.

Genomic Signatures from Clinical Tumor Sequencing in Patients with Breast Cancer Having Germline BRCA1/2 Mutation.

BRCA1 and BRCA2 are among the most important genes involved in DNA repair via homologous recombination (HR). Germline BRCA1/2 (gBRCA1/2)-related cancers have specific characteristics and treatment options but conducting gBRCA1/2 testing and interpreting the genetic imprint are sometimes complicated. Here, we describe the concordance of gBRCA1/2 derived from a panel of clinical tumor tissues using next-generation sequencing (NGS) and genetic aspects of tumors harboring gBRCA1/2 pathogenic variants. Targeted sequencing was performed using available tumor tissue from patients who underwent gBRCA1/2 testing. Comparative genomic analysis was performed according to gBRCA1/2 pathogenicity. A total of 321 patients who underwent gBRCA1/2 testing were screened, and 26 patients with gBRCA1/2 pathogenic (gBRCA1/2p) variants, eight patients with gBRCA1/2 variants of uncertain significance (VUS; gBRCA1/2v), and 43 patients with gBRCA1/2 wild-type (gBRCA1/2w) were included in analysis. Mutations in TP53 (49.4%) and PIK3CA (23.4%) were frequently detected in all samples. The number of single-nucleotide variants (SNVs) per tumor tissue was higher in the gBRCA1/2w group than that in the gBRCA1/2p group (14.81 vs. 18.86, p=0.278). Tumor mutation burden (TMB) was significantly higher in the gBRCA1/2w group than in the gBRCA1/2p group (10.21 vs. 13.47, p=0.017). Except for BRCA1/2, other HR-related genes were frequently mutated in patients with gBRCA1/2w. We demonstrated high sensitivity of gBRCA1/2 in tumors analyzed by NGS using a panel of tumor tissues. TMB value and aberration of non-BRCA1/2 HR-related genes differed significantly according to gBRCA1/2 pathogenicity in patients with breast cancer.

Genomic variation associated with carcinoma showing thymus‐like elements (CASTLE) in thyroid gland

BackgroundCarcinoma showing thymus‐like elements (CASTLE) is a rare kind of malignant tumor of thyroid gland. The genetic mutation characteristics of CASTLE are not clear.MethodsWe retrospectively analyzed seven patients diagnosed as CASTLE tumor in our hospital, and performed whole exome sequencing (WES) in five cases to analyze the genomic variation of CASTLE in thyroid gland.ResultsThe diagnosis of CASTLE was confirmed by histopathological and immunohistochemical results. Immunohistochemical staining showed that cell membranes of tumor samples in all cases were moderately to strongly positive for CD5 and CD117. WES presented a large number of single nucleotide variants (SNVs), insertions and deletions (InDel), and copy number variations (CNVs). By comparing with the TCGA database, we found novel mutations in significantly mutated genes such as FBXL16, PAQR7, LEFTY1, UBA52, and FLNA, as well as in potential disease‐related driver genes such as MLLT10, FLNA, CYLD, HLA‐B, KMT2D, SFPQ, MUC16, EEF2, and KMT2C. ConclusionsCASTLE tumors contain unique tumor driver gene mutations. The information about mutations in several novel genes obtained in this study may contribute to unraveling the molecular mechanisms responsible for the emergence of thyroid CASTLE tumors and help formulating possible in‐roads for treatment.

The Genomic Landscape of Corticotroph Tumors: From Silent Adenomas to ACTH-Secreting Carcinomas.

Corticotroph cells give rise to aggressive and rare pituitary neoplasms comprising ACTH-producing adenomas resulting in Cushing disease (CD), clinically silent ACTH adenomas (SCA), Crooke cell adenomas (CCA) and ACTH-producing carcinomas (CA). The molecular pathogenesis of these tumors is still poorly understood. To better understand the genomic landscape of all the lesions of the corticotroph lineage, we sequenced the whole exome of three SCA, one CCA, four ACTH-secreting PA causing CD, one corticotrophinoma occurring in a CD patient who developed Nelson syndrome after adrenalectomy and one patient with an ACTH-producing CA. The ACTH-producing CA was the lesion with the highest number of single nucleotide variants (SNV) in genes such as USP8, TP53, AURKA, EGFR, HSD3B1 and CDKN1A. The USP8 variant was found only in the ACTH-CA and in the corticotrophinoma occurring in a patient with Nelson syndrome. In CCA, SNV in TP53, EGFR, HSD3B1 and CDKN1A SNV were present. HSD3B1 and CDKN1A SNVs were present in all three SCA, whereas in two of these tumors SNV in TP53, AURKA and EGFR were found. None of the analyzed tumors showed SNV in USP48, BRAF, BRG1 or CABLES1. The amplification of 17q12 was found in all tumors, except for the ACTH-producing carcinoma. The four clinically functioning ACTH adenomas and the ACTH-CA shared the amplification of 10q11.22 and showed more copy-number variation (CNV) gains and single-nucleotide variations than the nonfunctioning tumors.

Distinct Genomic Profiles Are Associated with Treatment Response and Survival in Ovarian Cancer.

Simple SummaryIn most patients with ovarian cancer, their disease eventually becomes resistant to chemotherapy. The timing and type of treatment given are therefore highly important. Currently, treatment choice is mainly based on the subtype of cancer (from a histological point of view), prior response to chemotherapy, and the time it takes for the disease to recur. In this study, we combined complete genome data of the tumor with clinical data to better understand treatment responses. In total, 132 tumor samples were included, all from patients with disease that had spread beyond the primary location. By clustering the samples based on genetic characteristics, we have identified subgroups with distinct response rates and survival outcomes. We suggest that in the future, this data can be used to make more informed treatment choices for individuals with ovarian cancer.The majority of patients with ovarian cancer ultimately develop recurrent chemotherapy-resistant disease. Treatment stratification is mainly based on histological subtype and stage, prior response to platinum-based chemotherapy, and time to recurrent disease. Here, we integrated clinical treatment, treatment response, and survival data with whole-genome sequencing profiles of 132 solid tumor biopsies of metastatic epithelial ovarian cancer to explore genome-informed stratification opportunities. Samples from primary and recurrent disease harbored comparable numbers of single nucleotide variants and structural variants. Mutational signatures represented platinum exposure, homologous recombination deficiency, and aging. Unsupervised hierarchical clustering based on genomic input data identified specific ovarian cancer subgroups, characterized by homologous recombination deficiency, genome stability, and duplications. The clusters exhibited distinct response rates and survival probabilities which could thus potentially be used for genome-informed therapy stratification for more personalized ovarian cancer treatment.

Abstract PD14-04: Circulating tumor DNA characterization of invasive lobular carcinoma in patients with metastatic breast cancer

Abstract Background:Invasive lobular carcinoma (ILC) is the second most common histology of breast cancer, accounting for approximately 10-15% of cases. Prior studies have demonstrated that loss of E-cadherin, as well as alterations in tissue including CDH1, FOXA1, TBX3 and PTEN loss, that were more commonly observed in Luminal A ILC, while GATA3 was more commonly observed in invasive ductal carcinoma (IDC) (Ciriello et al., Cell 2015). However, data regarding the characterization of circulating tumor DNA (ctDNA) in patients (pts) with metastatic ILC are limited. We hypothesized that there would be distinct mutational profiles between pts with metastatic ILC and IDC that could be characterized using ctDNA. Methods:This retrospective cohort study included de-identified clinical, pathological, and ctDNA data from pts with metastatic breast cancer (MBC) combined under a data use agreement and approved by the institutional review boards of three sites: Washington University in St. Louis (MO), Northwestern University (Chicago, IL), and Massachusetts General Hospital (Boston, MA). All pts included in the study had ctDNA testing per standard of care with plasma-based genotyping performed by Guardant360 (Redwood City, CA) between 2015-2020. Histological classification (ILC vs. IDC) was defined based on review of pathology reports from the primary tumor or from breast biopsies of de novo MBC, and additional clinical and pathological variables were obtained via electronic medical record review. Single nucleotide variants (SNVs) were annotated using OncoKB and ClinVar and only pathogenic variants were included. Mutational profiles were compared across histologic subtypes using Fisher’s exact test to assess differences in alteration frequency across subtypes. Multivariable analysis was performed. Results:A total of 994 pts with MBC underwent ctDNA testing and were included in the analysis. 10.7% of pts had ILC (N=106) and 89.3% had IDC (N=888). 89.4% of ILC cases were categorized as hormone-receptor positive (HR+) compared with 67.1% of IDC cases. Pts with ILC had a lower frequency of triple-negative (6.7% vs. 17.7%) and HER2 positive (3.9% vs. 15.2%) breast cancer compared with IDC. Pts with ILC had a significantly higher number of pathogenic SNVs compared with IDC (mean 4.45 vs. 2.77; P=0.0037). In contrast, pts with ILC had a significantly lower number of copy number alterations as compared to pts with IDC (mean 0.40 vs. 1.03; P=0.0017). No differences were observed in mutant allele frequency between pts with ILC and IDC. The 5 most common alterations observed in pts with ILC were the following: PIK3CA, TP53, ESR1, ERBB2, and ARID1A. Alterations in AR, BRAF, CDH1, ERBB2, FGFR2, IDH2, KRAS, NF1, PIK3CA, SMAD4, and TERT were significantly higher in ILC than IDC (all P<0.05). In contrast, mutations in GATA3, and amplifications in ERBB2 and MYC were significantly more common in pts with IDC (all P<0.05). In multivariable analysis, mutations in BRAF, CDH1, ERBB2, IDH2, TERT remained significantly higher in ILC, while amplification of MYC was significantly higher in IDC (all P<0.05). After restricting the analysis to pts with HR+ HER2 negative MBC, the following genes were significant in multivariate analysis: CDH1 and ERBB2 for pts with ILC and MYC amplification for pts with IDC (all P<0.05). Discussion:In this large, multi-institutional dataset, pts with metastatic ILC were characterized by a significantly higher number of SNVs in ctDNA compared to pts with IDC, suggesting higher mutational burden. We report several alterations that were significantly different in ILC vs. IDC. These results demonstrate the ctDNA profile of pts with ILC, and future studies should explore serial plasma-based genotyping to track ILC evolution to develop targeted precision medicine based therapeutic approaches for this unique subset of pts with MBC. Citation Format: Andrew A Davis, Lorenzo Gerratana, Katherine Clifton, Marko Velimirovic, Whitney L Hensing, Ami N Shah, Paolo D’Amico, Carolina Reduzzi, Qiang Zhang, Charles S Dai, Elyssa N Denault, Nusayba A Bagegni, Mateusz Opyrchal, Foluso O Ademuyiwa, Ron Bose, William J Gradishar, Amir Behdad, Cynthia X Ma, Aditya Bardia, Massimo Cristofanilli. Circulating tumor DNA characterization of invasive lobular carcinoma in patients with metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-04.