Target Enrichment Research Articles

Target enrichment improves culture-independent detection of Neisseria gonorrhoeae and antimicrobial resistance determinants direct from clinical samples with Nanopore sequencing.

Multi-drug-resistant Neisseria gonorrhoeae infection is a significant public health risk. Rapidly detecting N. gonorrhoeae and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of N. gonorrhoeae DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire N. gonorrhoeae genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology.

High-throughput sequencing (HTS) has revolutionised the field of pathogen genomics, enabling the direct recovery of pathogen genomes from clinical and environmental samples. However, pathogen nucleic acids are often overwhelmed by those of the host, requiring deep metagenomic sequencing to recover sufficient sequences for downstream analyses (e.g., identification and genome characterisation). To circumvent this, hybrid-capture target enrichment (HC) is able to enrich pathogen nucleic acids across multiple scales of divergences and taxa, depending on the panel used. In this review, we outline the applications of HC in human pathogens-bacteria, fungi, parasites and viruses-including identification, genomic epidemiology, antimicrobial resistance genotyping, and evolution. Importantly, we explored the applicability of HC to clinical metagenomics, which ultimately requires more work before it is a reliable and accurate tool for clinical diagnosis. Relatedly, the utility of HC was exemplified by COVID-19, which was used as a case study to illustrate the maturity of HC for recovering pathogen sequences. As we unravel the origins of COVID-19, zoonoses remain more relevant than ever. Therefore, the role of HC in biosurveillance studies is also highlighted in this review, which is critical in preparing us for the next pandemic. We also found that while HC is a popular tool to study viruses, it remains underutilised in parasites and fungi and, to a lesser extent, bacteria. Finally, weevaluated the future of HC with respect to bait design in the eukaryotic groups and the prospect of combining HC with long-read HTS.

Abstract 3656: Target enrichment of methylated circulating tumor DNA for colorectal cancer detection

Abstract Aberrant DNA methylation changes are one of the earliest signatures of cancer development. Methylation profiling of circulating tumor DNA (ctDNA) from plasma has emerged as a promising approach for early cancer detection (ECD). However, the set of methylation targets that can successfully distinguish cancer through liquid biopsy will vary depending on cancer type and subtype. Here, we explored a methylation target enrichment workflow and developed a methylation-based classification model to distinguish between patients with colorectal cancer (CRC) and healthy individuals. First, CRC-specific CpG targets were identified by comparing the methylation landscape of CRC and normal samples (tissue and blood) available in The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) datasets. This analysis resulted in >800 CRC-specific CpG targets for initial evaluation. To develop and test the classification model, CRC patients and healthy individuals with plasma samples were included. A machine learning model was used to evaluate the highest performing CpG targets that effectively discriminated between CRC patients with detected ctDNA (“CRC ctDNA-positive”) and healthy individuals. Correlation between methylation levels of the high performing CpG targets and the variant allele frequency (VAF) among CRC ctDNA-positive patients was calculated. In total, 86 patients were included in this proof of concept study (mean age, 62 ±12 years; male, 55%). 50 CRC ctDNA-positive patients (24% stage I, 40% stage II, 24% stage III, and 12% stage IV) and 36 healthy normals were included in the analysis. We investigated this cohort to identify the highest performing CpG targets among the >800 potential CpG targets that provided effective discrimination between CRC patients and healthy individuals. Using the highest performing CpG targets, observed methylation level was correlated with VAF of single nucleotide variants detected by ctDNA testing (R2: 0.8). In summary, we developed a cost-effective methylation target enrichment workflow and utilized machine-learning algorithms to demonstrate the potential use of ctDNA methylation markers in early detection. Additional studies are needed to confirm that these and other markers can accurately distinguish between patients with CRC and healthy individuals. Citation Format: Hsiao-Yun Huang, Ravi Vijaya Satya, Wen-Ching Chan, Esha Atolia, Joshua Babiarz, Bernhard Zimmermann, Trupti Kawli. Target enrichment of methylated circulating tumor DNA for colorectal cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3656.

Abstract 320: Development and automation of a streamlined targeted enrichment method for cancer mutation detection

Abstract Next Generation Sequencing (NGS) of DNA is a powerful tool for the detection of genetic variations, including single nucleotide polymorphisms, copy number variation, and small insertions/deletions. Target enrichment technologies enhance DNA sequencing by enabling users to analyze specific regions of interest only, which helps to effectively increase sequencing depth and sample throughput while minimizing cost. However, many targeted DNA library construction workflows are not optimal for routine genetic testing needs due to inconsistent, lengthy workflow. This poses significant challenge to application like cancer mutation profiling that requires detection sensitivity down to 1% or below due to heterogeneous nature of tumor tissue. In addition, high throughput testing with minimal hands on time is desirable with the rapid advance of precision medicine. Magnetic bead separations are commonly used between the individual reactions of targeted DNA library preparation workflows for cleanup and size selection. These bead clean up reactions are not only labor-intensive and tedious procedures but can also greatly contribute to sample-to sample variation especially when employing automated library preparation procedures. With the sophisticated QIAseq Targeted DNA Pro, we developed a novel streamlined targeted DNA workflow that greatly reduces handling variation by replacing the majority of bead clean up steps with enzymatic reactions, which now can be finished in as little as six hours. In combination with the newly developed QIAseq Normalizer kit, the protocol has been successfully implemented on the Hamilton NGS Star liquid handling platform resulting in up to 96 highly uniform, complex and ready-to-sequence libraries that can be effortless generated in one run. This complete automated solution saves researcher up to four hours of hands-on time. When using commercial reference samples, cancer mutations down to 1% were consistently detected with this automated workflow. The presented data demonstrate that this integrated and streamlined workflow does not only provide an automation friendly workflow but also allows to reliably identify low frequent cancer mutations. The applications presented here are for research use only. Not for use in diagnostic procedures. Citation Format: Zhong Wu, Michelle Baird, Cornelia Mechlen, Peter Hahn, Jonathan M. Shaffer. Development and automation of a streamlined targeted enrichment method for cancer mutation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 320.

Abstract 6085: Leveraging targeted epigenetic and genetic detection for cost-effective cancer classification

Abstract The combinatorial power of genetics and epigenetics is vital to understanding biology in healthy and cancerous states. Utilizing a recent novel approach, we enable the simultaneous identification of modified cytosine and the canonical bases A, C, T & G in an enzymatic single-workflow solution. Generating this information across the whole genome provides a much-improved understanding of genetic and epigenetic changes, but can require a large amount of sequencing, particularly when exploring at depth. Enrichment protocols such as target enrichment enable deep interrogation of target areas whilst maintaining cost effectiveness. Target enrichment panels enable the selective isolation of sequences of interest through target hybridization. Using commercially available panels combined with the novel enzymatic single-workflow solution we show uniform performance across a range of input amounts with reduced sequencing requirements. Samples used were cell free DNA (cfDNA) from patient samples with either Stage I or IV Colorectal Cancer and reportedly healthy controls and were targeted using pan-cancer and methylome panels. Widespread differences such as hypermethylation are clearly observed in late-stage cancer vs early-stage or healthy controls demonstrating utility of the approach for applications such as liquid biopsy. Refining genetic and epigenetic investigations to a narrower region of interest can improve detection of low prevalence disease associated biomarkers at significant sequencing depth and lower sequencing costs. Citation Format: Robert Crawford, Ben Krajacich, Ermira Lleshi, Aurel Negrea, Lisabet Andreasen, Jens Fullgrabe, Jack Monahan, David Morley, Fabio Puddu, Donna McDade Walker, Semyon Kruglyak, Páidí Creed. Leveraging targeted epigenetic and genetic detection for cost-effective cancer classification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6085.

Abstract 2935: NGS panel customization for comprehensive genomic profiling

Abstract Introduction: We have developed a custom SureSelect Cancer design pipeline for the creation of target enrichment gene panels for comprehensive profiling of single nucleotide variations (SNP/INDEL), gene-level copy number variations (CNV), and DNA translocations (TL) as well as content for measuring of microsatellite instability (MSI) and tumor mutational burden (TMB). Methods: The performance of individual probes was measured for the 679-gene SureSelect Cancer CGP panel and the information used to generate a reference database. When creating a new design, the gene list is cross-referenced with the database and when applicable, the probe information is applied to the new design. If genes are included in the custom design that are not present in the database, a second design algorithm selects the optimal probe based on its sequence characteristics (% GC, homology, etc.). Content is selected based on the biomarker to be measured and can be defined based on gene name (SNV/indel and CNV) or genomic coordinates (SNV/indel, TL). Pre-defined content for MSI measurement can be added to any design to cover 288 microsatellite sites. The performance of these sites in determining MSI status was demonstrated by comparing to the MSI-PCR reference method. To assess the performance of the design methodology, designs of various sizes (50 to 650+ genes) and content type were generated and assayed on reference samples and FFPE with known biomarkers. Sequencing metrics were evaluated for capture specificity and coverage of the target regions. Results: The design methodology allowed for sub-selection of content present in the 679-gene SureSelect Cancer CGP assay as well as addition of de-novo content. The design algorithm uses empirical data on probe performance to provide robust and reproducible results for the range of supported design sizes. Biomarkers were reliably detected with SNV detection down to 1% allele frequency, detection of gene amplifications and translocations concordant with FISH, and MSI and TMB determination concordant with PCR and Exome sequencing respectively. The design workspace was implemented in the SureDesign platform, providing a web-based interface for creating the designs. Conclusions: We demonstrated full customization capability of the SureSelect Cancer target enrichment platform that pairs user-defined content with an optimized probe design methodology to enable the sensitive detection of the common types of DNA alterations found in FFPE samples. Citation Format: Michael Ruvolo, Akanksha Khare, Nishita Matcha-Balaguruvappa, Katherine Wilkins, Linus Forsmark, Swati Gupta. NGS panel customization for comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2935.

Abstract 6274: Integrated multi-omics revealed the involvement of MYC and mTOR pathways in radiation resistance of adrenocortical cancer

Abstract The morbidity and mortality of patients with adrenocortical cancer (ACC) are commonly due to locoregional postoperative recurrence and progressive systemic metastasis. Surgical resection remains the only curative treatment option, but even with complete resection, locoregional recurrence rates remain high. Adjuvant radiation therapy (RT) has been shown to reduce recurrence rates and may improve overall survival in selected patients. However, half of the patients receiving postoperative RT still develop locoregional recurrence, clearly demonstrating the heterogeneous nature of ACC. Minimal investigation into the mechanisms underlying radiation response and resistance in ACC has been performed, but elucidating this information is critical to improving outcomes. To address this knowledge gap, NCI-H295R cells were treated with fractionated radiation at 1 Gray (Gy) per day for 5 consecutive days for two consecutive weeks, cumulating in a total radiation dose of 10 Gy. Treated and control ACC cells were harvested on days 7 and 14 for bulk RNA-sequencing (RNA-Seq) and total proteomics by liquid chromatography and tandem mass spectrometry. Significantly differentially expressed genes (DEGs) and proteomes (adj. p. value <0.05) that overlapped between day 7 and day 14 were selected for further analysis and comparison with publicly available gene expression data sets in ACC (The Cancer Genome Atlas (TCGA); Gene Expression Omnibus: GSE12368, GSE33371, GSE75415, GSE90713). There were 3993 DEGs and 423 proteomes identified, respectively. Of these, there were 139 overlapping DEGs with respective proteomes found, 72 of which were differentially expressed in the 4 GEO data sets comparing normal adrenal cortex to ACC. RNA-Seq pathway analysis demonstrated enrichment of E2F targets, G2M checkpoint, mTORC1 signaling, and MYC targets v1, while total proteomics pathway analysis revealed enrichment of cell cycle, transcriptional regulation, cellular stress and injury, alterations of RNA metabolism, and metabolism proteomes. Given that MYC and mTOR are both involved in transcriptional regulation and cell cycle regulation, and mTORC1 in particular is associated with cellular metabolism, the RNA-Seq and proteomics pathway analyses show general consistency with each other. TCGA gene expression analysis showed that overexpression of MYC or mTOR was correlated with shorter overall survival (OS) and disease-free survival (DFS) in the entire ACC cohort (p<0.05). Together, the combination of in vitro pathway analysis and TCGA analysis suggests a potential involvement of the MYC and mTOR pathways in radiation resistance in ACC. Further in vitro and in vivo studies are warranted to identify specific molecular compounds that target MYC and mTOR pathways and improve radiation response in ACC. Citation Format: Bhavishya Ramamoorthy, Prachi Mishra, Myriem Boufraqech, Nicholas Michael, Margaret Cam, Richard Finney, Thomas Meyer, Ronald Holewinski, Thorkell Andresson, Madeline Wong, Steven Shema, Desiree Tillo, Naris Nilubol. Integrated multi-omics revealed the involvement of MYC and mTOR pathways in radiation resistance of adrenocortical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6274.

Abstract 5049: Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost

Abstract We aimed to develop a single streamlined assay to detect all Glioma biomarkers. The current diagnosis of Gliomas incorporates molecular biomarkers that include whole chromosomal copy number variations (CNVs) and single nucleotide variations (SNVs). Testing CNVs and SNVs are traditionally done using different large platforms and are performed in large genomic facilities that have the suitable infrastructure. This creates delays in testing, where samples undergo lengthy referrals and the tissue is often split between institutions, which also adds substantially to the costs. We have previously used Nanopore sequencing to develop an assay to detect IDH SNVs from formalin-fixed paraffin-embedded (FFPE) tissue. Nanopore sequencing platform uses small inexpensive tools and can test for both CNVs and SNVs. However, combining both targets efficiently in FFPE tissue remains a challenge. While nanopore whole genome sequencing can detect CNVs, it does not allow for enough coverage to accurately detect small SNVs. Also, raw FFPE DNA is not compatible with the nanopore technology. To fix both challenges we used a PCR based approach that allows enrichment of the SNV targets, as well as creates new nanopore compatible DNA copies. To counteract the bias that PCR creates with uneven amplification, we used a SNP based method to call chromosomal gains and losses. Statistical analysis of probability showed that targeting 15 heterozygous SNPs per chr arm is sufficient for accurate CNV detection (error rate <0.1%). A total of 57 amplicons targeting CNVs on Chr 1,7, 10, 19, and EGFR gene; and IDH1 and IDH2 SNVs were pooled together in one assay and tested on 4 Glioma samples. Variant calling was performed with a custom Python script. All CNV targets were accurately detected on all samples. Losses in chr 1p, 19q and chr 10 were evident with a loss of heterozygosity (LOH) pattern on the SNP analysis. The gain in chromosome 7 was also observed as an allele gain 2:1 SNP pattern on the corresponding samples. The SNV statuses of these samples were also accurately detected when compared to reference results. Finally, the SNV and CNV calling analysis were streamlined using custom shell script, further reducing total turnaround time (including sequencing and data analysis) to less than a day for the entire batch of samples. Initial cost analysis shows <50% of the traditional testing costs for the assay. This work is the first to develop a streamlined single test to detect CNVs and SNVs in Gliomas using FFPE DNA on the nanopore sequencing platform. The shorter testing time, streamlined workflow, and low assay/capital cost could positively influence the care of brain cancer patients. Notably, nanopore-based sequencing tools can also be implemented in other types of tumor diagnosis, especially by smaller labs, helping to overcome many of the existing diagnostic challenges. Citation Format: Mashiat L. Mimosa, Jared T. Simpson, Karel Boissinot, Mora Tiab, Ramzi Fattouh, Rola M. Saleeb. Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5049.

Abstract 7065: AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer, with emerging evidence highlighting the role of cancer stem cells (CSCs) in its progression. AMBRA1 has been implicated to exert both tumor-promoting and tumor-suppressive roles through regulating cell cycle and mitophagy in various solid tumors. However, the functional roles of AMBRA1 in modulating cancer stemness and tumorigenesis in HCC remain elusive. Initial analysis of The Cancer Genome Atlas data revealed a significant inverse correlation between AMBRA1 expression and stemness indices (TSS) in HCC patients, with low AMBRA1 levels associated with an upregulated oxidative phosphorylation (OXPHOS) gene signature. To elucidate the functional consequences of AMBRA1 modulation in liver tumor development and stemness maintenance, we exploited both hydrodynamic mouse models and human HCC cell lines. Our findings indicate that hepatic AMBRA1 deletion enhances cancer stemness and tumorigenesis both in vivo and in vitro. Moreover, HCC cells with reduced AMBRA1 expression demonstrated heightened mitochondrial activity, characterized by increased oxygen consumption, respiration, mitochondrial membrane potential, and intracellular ATP levels. Apart from OXPHOS, we also observed a concomitant up-regulation and enrichment of MYC targets in TSS-high and AMBRA1-low HCC patients, pointing towards a deregulated c-MYC signaling to promote cancer stemness under AMBRA1 regulation in HCC. Collectively, our results demonstrate that AMBRA1 loss enhances mitochondrial function to support hepatic tumorigenesis in HCC. Citation Format: Yan Liu, Yunong Xie, Yimiao He, Stephanie Ma, Man Tong. AMBRA1 coordinates mitochondrial function to regulate cancer stemness and tumorigenesis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7065.

Abstract 2466: tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma

Abstract Background: Despite significant advances in immunotherapy-based treatment options for Head and Neck Squamous Cell Carcinoma (HNSCC), patient prognosis and survival remains poor. To enhance response to immune-based therapies, specific targeting of immunomodulatory pathways has been investigated through co-stimulatory gene targets, the microbiome, and more. An emerging biomolecule is Transfer RNAs (tRNAs), which are a class of RNAs that transport amino acids from the cytoplasm to ribosomes, and synthesize new proteins encoded by mRNA transcripts. tRNA-derived fragments (tRFs) are a category of cleaved, mature tRNA that participate in post-transcriptional regulation and have been implicated in the modulation of cancer and other immune-associated diseases. While tRFs are primarily implicated in diagnostic signatures for various cancers, few studies have investigated their role in modulating the immunotherapy-based response in HNSCC. In this study, we characterized the role of tRFs in modulating immunotherapy gene targets pertinent to HNSCC. Methods: We extracted tRF read counts from MINTbase 2.0 for 455 primary HNSCC tumor samples and 44 adjacent normals. Differential expression analysis was first performed to identify tRFs implicated in HNSCC tumors, with respect to adjacent normal tissue. Next, these tRFs were associated with immunotherapy target genes and response markers with Spearman’s correlation. Further characterization of these tRFs was performed by correlating their abundance to infiltrating immune cells, pathway enrichment of immunotherapy targets, and clinical variables. Results: Significant differential expression of tRFs were found between HNSCC primary tumor samples and adjacent normal tissue, including 7 tRFs (FDR < 0.05, |log fold change| > 0.5). All were enriched in normal samples. AlaCGC5tRF appeared to be an important regulator of immunotherapy target genes through increased co-expression of PTPN11, STAT3, and PTEN (p-value < 0.05, R > 0) and decreased co-expression of CD58 and TNFRSF14 (p-value < 0.05, R < 0). Immune Checkpoint Inhibitor (ICI) genes were significantly correlated with several tRFs. Among several pertinent tRF markers, AlaCGC5tRF was strongly associated with negatively correlated with infiltrating activated NK cells naïve CD4 T cells (p-value < 0.05, R < 0). Conclusion: Our study is the most comprehensive to date to characterize the role of tRF elements in modulating HNSCC immunotherapy targets. We found several potential tRF biomarkers that may be indicative of enhanced immunotherapy treatment susceptibility, including AlaCGC5tRF and ArgTCT3tRf. Further studies are needed to investigate how these tRFs may be targeted alongside immunotherapies to enhance response to treatment. Citation Format: Rishabh Yalamarty, Daniel John, Annie Do, Tianyi Chen, Megan Ngo, Pranava Gande, Matthew Uzelac, Wei Tse Li, Weg M. Ongkeko. tRNA-derived fragments as immunotherapy response targets in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2466.

Abstract 5771: Utilizing a photoreactive chloroalkane capture tag for target deconvolution

Abstract Identifying the physiologically relevant targets of bioactive compounds is often the rate limiting step toward understanding their mechanism of action. To this end, we developed a streamlined chemical proteomic strategy that utilizes a novel photoreactive cleavable chloroalkane tag, which can be attached to bioactive compounds to isolate their respective cellular targets for identification by mass spectrometry. The tag does not significantly affect compounds potency and membrane permeability, allowing for binding interactions to be established within intact cells under appropriate physiological conditions. UV-induced covalent photo-crosslinking “freezes” the cellular interactions and prevents their dissociation upon cell lysis. Effective enrichment of the cellular targets is achieved through their selective covalent capture onto HaloTag coated particles and subsequent selective release by tag cleavage. Putative targets identified by mass spectrometry can be readily verified using resonance energy transfer. By exchanging the chloroalkane tag for a fluorophore, direct binding to the targets can be revealed through proximity of the fluorophore to a small NanoLuc luciferase genetically fused to the putative target. This approach was used to identify physiologically relevant targets for bioactive compounds including those with low affinity and/or low abundance as well as insoluble targets with multiple trans membrane domains. Citation Format: Tetsuo Uyeda, Robin Hurst, Sergiy Levin, Mike Rosenblatt, Kris Zimmermann, Rachel F. Ohana. Utilizing a photoreactive chloroalkane capture tag for target deconvolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5771.

Abstract 321: Optimized RNA Seq library preparation for fusion calling from FFPE samples

Abstract Gene fusions due to chromosomal rearrangement, duplication, or deletion can be important drivers of cancer. Detection of gene fusions plays a valuable role in selecting targeted therapies. A widely used method for detecting fusion transcripts is targeted RNA sequencing, which focuses sequencing reads on known fusions but does not allow for the detection of novel fusions. Whole transcriptome analysis (WTA), where the entire transcriptome is interrogated, offers an opportunity to detect both known and novel fusions. Herein we examine the effect of sample quality, input mass, increased reverse transcriptase (RT) and insert size on fusion calling. Libraries are prepared using the Watchmaker RNA Library Preparation Kit with or without Polaris Depletion and, for target enrichment, the Twist Exome 2.0 panel prior to paired end 2 × 150 Illumina sequencing. First, control samples, specifically Seraseq Fusion RNA v4 high quality and FFPE samples, are used to determine if any of the parameters interrogated increase the number of breakpoint spanning reads both in a WTA and targeted sequencing context. Once established, the optimized workflow is applied to real FFPE samples. Targeted sequencing results in a higher number of breakpoint spanning reads, compared to WTA, when controlling for sequencing depth, resulting in a higher number of reads supporting each fusion call. Longer insert sizes, achieved by modified SPRI ratios, maintain library complexity and may enhance fusion calling. Taken together, these data demonstrate an optimized protocol for fusion calling from FFPE and demonstrate the utility of both WTA and targeted sequencing for fusion detection. Citation Format: Giulia Corbet, Josh Haimes, Travis Sanders, Martin Ranik, Thomas Harrison, Eduard Casas, Kailee Reed, Doug Wendel, Kristina Giorda, Ariele Hanek, Jen Pavlica, Brian Kudlow. Optimized RNA Seq library preparation for fusion calling from FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 321.

Abstract 4626: A kit targeting detection of ESR1 mutations from circulating exosomal RNA and cell-free DNA supports longitudinal studies into endocrine therapy resistance in a broadly accessible RT-qPCR format

Abstract Introduction: The most prevalent subtype of breast cancer is HR+/HER2- (positive for hormone receptor, negative for human epidermal growth factor receptor 2). For metastatic breast cancer (mBC), a frequent occurrence is resistance to the aromatase inhibitors used in endocrine therapy (ET). Resistance is mediated through mutations in the ligand binding domain of estrogen receptor 1 (ESR1). Approval of elacestrant, a second-line agent for HR+/HER2-/ESR1 mutated mBC , underscores the need for early detection of ESR1 resistance mutations. We have developed sensitive RT-qPCR reagents for targeted longitudinal studies into the emergence of ESR1 resistance mutations in plasma. This all-inclusive kit analyzes circulating exosomal RNA (exoRNA) and cell-free DNA (cfDNA) to accurately identify low copy number mutations with a rapid turnaround time. Methods: Challenge panels were constructed using blends of synthetic nucleic acids of known mutational status in a background of partially fragmented wild-type DNA from cell lines. Additionally, plasma samples collected from subjects with stage IV mBC (HR+/HER2-) on active aromatase inhibitor therapy, +/- CDK 4/6 inhibitor for a minimum of one year, were subjected to an in-house method optimized to co-enrich exoRNA and cfDNA. Multiplex RT-qPCR-based target enrichment performed on 11 ESR1 mutations and an internal control were evaluated on widely used thermal cycler and qPCR instruments. Results: We developed technology that interrogates 11 key ESR1 mutations associated with ET resistance in HR+/HER2- mBC cases. Earlier studies performed in simplex analyzed plasmid-based ESR1 mutations with detection of 10 or fewer mutant copies, estimating analytical sensitivity of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Herein, we describe the subsequent work to complete the methodology employed in the kit, including the incorporation of an endogenous control, external batch run control materials (positive, negative), deeply optimized reagents (RT, pre-amplification, qPCR), and automated analysis. We report on the results of studies into the kit’s performance characteristics against their targets: analytical sensitivity of the equivalent of 5 copies of mutation per mL plasma, high precision across replicates at 0.1% allele fraction, analytical specificity of ≥90% negative results in mutation-negative replicates, as well as workflow attributes (reduced pipetting steps and turnaround time). Conclusion: Our solution to detect ESR1 resistance mutations facilitates the generation of nucleic acid sample to results within one day using liquid biopsy samples and a multiplexed RT-qPCR. Enhancing mutation detection sensitivity by complementing cfDNA with exoRNA will facilitate future research into breast cancer treatment options. Citation Format: Justin T. Brown, Julie R. Thibert, Liangjing Chen, Melissa Church, Holli Dale, Jamie Myers, Elliot Hallmark, Megan Yociss, J Aquiles Sanchez, Kurt Franzen, Johan Skog, Gary Latham, Brian Haynes, Sarah Statt. A kit targeting detection of ESR1 mutations from circulating exosomal RNA and cell-free DNA supports longitudinal studies into endocrine therapy resistance in a broadly accessible RT-qPCR format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4626.

Abstract 1029: Role of PCDHGA12/PRRX1 methylation for the diagnosis of lung cancer using bronchial washing fluid

Abstract Bronchoscopy is a frequently used initial diagnostic procedure for patients with suspected lung cancer (LC). Cytological examinations of bronchial washing (BW) samples, obtained during bronchoscopy, often yield inconclusive results in the diagnosis of LC. Aberrant DNA methylation has previously been shown to be a useful biomarker for LC. DNA methylation analysis in BW specimens could serve as a valuable clinical adjunct to cytology. In this study, to identify potential methylation biomarkers for LC, methylation microarray analyses were performed on primary tumors and compared with adjacent non-tumor tissues of 13 patients with LC at various stages (I - IV). A stepwise validation process using bisulfite-pyrosequencing of cell lines and tissues identified six methylated genes (ADAMTS20, FOXC2, NKX2-5, OLIG3, PCDHGA12, and PRRX1) associated with LC. To assess methylation in BW samples, a highly sensitive and accurate 3-plex Linear Target Enrichment (LTE)-quantitative methylation-specific PCR (qMSP) in a single closed tube was developed. Clinical validation was performed using BW samples from patients with LC (n = 68) and non-LC (benign) (n = 33) and identified PCDHGA12 and PRRX1 methylation as the best performing marker combination for detecting LC. The 2-marker combination exhibited a sensitivity of 82.4% and a specificity of 87.9%, with an area under the curve of 0.891. Notably, its sensitivity was 100% for small cell LC. The 2-marker combination had a positive predictive value of 93.3% and a negative predictive value of 70.7%. Its sensitivity outperformed cytology. The methylation status of the 2-marker showed no correlation with gender, age or stage, but was associated with tumor location and histology. In conclusion, we identified PCDHGA12 and PRRX1 methylation as high potential biomarkers for LC diagnosis. This study shows that these biomarkers could be used as a diagnostic adjunct to cytology in the clinical practice for diagnosing LC in BW specimens. Citation Format: Ji Woong Son, Tae Jeong Oh, Minheyok Lee, Chang Ryul Park, Sungwhan An. Role of PCDHGA12/PRRX1 methylation for the diagnosis of lung cancer using bronchial washing fluid [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1029.

Abstract 6255: Personalized medicine for DCIS

Abstract Background: In 2023, 55,720 women will be diagnosed with non-invasive ductal carcinoma in situ (DCIS) in the United States. We evaluated RNA sequencing (RNA-Seq), clinical and social factors that drive subsequent breast events in a DCIS study population enriched for Black women (30%). Methods: RNA was extracted from macrosections of archival formalin-fixed paraffin-embedded (FFPE) DCIS tissue from Resource Archival Human Breast Tissue (RAHBT) from the St. Louis Breast Tissue Registry at Washington University School of Medicine and the Dana Farber Cancer Institute patient cohorts. Tissues were prepared by microsection of 5 uM whole section from unstained slides (Fisher). RNA was extracted using Qiagen RNeasy FFPE Kits from macrosectioned DCIS specimens. Purified total RNA was evaluated for quality and quantity using the Agilent Fragment Analyzer RNA assay. Bulk RNA sequencing was piloted in DCIS case-control samples using the SMART-seq and Hybrid Capture platforms at the DFCI Molecular Biology Core Facilities. Raw reads were aligned to the GRCh38/hg38 reference genome using STAR. Preliminary quality control analysis of a curated gene list showed higher quality data using the Hybrid Capture technology. Libraries for Hybrid Capture were pooled in 8plex reactions and hybridized to Twist Exome 2.0 probes for target enrichment. The final library pools were sequenced on an Illumina NovaSeq. Cases were defined as women diagnosed with DCIS in 1999 or later with subsequent invasive breast cancer, local DCIS recurrence, or lobular CIS recurrence. Controls were defined as women diagnosed with DCIS in 1999 or later without subsequent breast events during a similar follow-up time. The R package DESeq2 was used for data analysis. Results: The mean age at DCIS diagnosis was 56.3 (Std Dev 10.23). The mean follow-up time was 147.8 months. Hybrid Capture RNA-Seq files are stored and analyzed from the Harvard Medical School O2 Cluster using R programming (version 4.2.2, DEGreport). Quality control analyses demonstrated that the majority of the samples sequenced by Hybrid Capture had over 20 million total reads. Principal Component Analyses (PCA) and hierarchical clustering analyses of 19,821 protein-coding genes were conducted on the 143 DCIS samples sequenced using Hybrid Capture. PC1 was race (9%). A significant association was observed for follow-up time (PC3, 4.03%). First, we explored the association of the candidate gene ERBB2. The mean transcript abundance for ERBB2 was 11784.37 (case-control p-value=0.08). Preliminary analysis of selected genes of interest did not show statistically significant differences between cases and controls after FDR p-value adjustment. Differential gene expression analyses are ongoing. Conclusion: This study demonstrates the feasibility of generating high-quality RNA-seq data from diverse, archival DCIS samples. Citation Format: Aditi Hazra. Personalized medicine for DCIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6255.

Abstract 5054: Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications

Abstract Formalin-fixed paraffin-embedded (FFPE) biopsies are highly valuable and widely used tissue specimens for clinical diagnostics. However, obtaining sufficient and high-quality nucleic acid material from limited FFPE samples presents a challenge for downstream molecular analysis, such as next-generation sequencing (NGS). We present an optimized sequential extraction method that generates high-quality DNA and RNA from a single set of input tissues that is automatable and operation-friendly. This workflow performs well with reduced FFPE tissue input and efficiently supports various high-throughput clinical NGS applications. The DNA/RNA yield, quality, purity, and impacts on NGS assay performances were on par or better than an existing validated comparator extraction method. With comparable FFPE input, the new method demonstrated a superior extraction performance with significantly higher yield, quality, and purity. For DNA, NGS libraries were made with two different library preparation methods: a TA-ligation-based method and a single-strand-based method, followed by hybrid capturing and sequencing. The DNA from the new extraction method demonstrated a superior library conversion rate and improved target enrichment uniformity with both chemistries. This provides the potential of reduced input requirements, allowing very limited tissue, such as fine needle aspirate or core needle biopsy, for clinical NGS testing. The sequencing results were highly concordant between the existing and new extraction methods when the extracted DNA was subjected to a validated comprehensive genomic profiling (CGP) clinical test, demonstrating the quality and robustness of the extraction. For RNA, NGS libraries were made following the validated CGP clinical test with hybrid capturing, and the results were comparable between the two extraction methods. In conclusion, an FFPE extraction method was optimized to allow more clinical biopsy samples to be tested with different NGS workflows, providing a better diagnostic value for patient care. Citation Format: Rebecca Hong, Jieying Chu, Steven Hsiao, Daniel Whang, Steven P. Rivera, Heng Xie, Jiannan Guo. Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5054.

Abstract 7507: Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients

Abstract Background: Single circulating tumor cells (sCTC) capture without cell fixation with no blood contaminants like leukocytes are highly challenging. sCTC genome transcriptomics anticipates new avenues in new discovery, targeted therapies, and inclusion of patients in clinical trials where the tissue is inaccessible. We report, an assay to selectively capture live sCTCs at singularity with NGS-based comprehensive mutational profiling in breast cancer (BC) patients. Further, paired ctDNA outcomes using blood with mutational heterogeneity scores of sCTCs adds comprehensive genomics for establishing an accelerated treatment outcomes and resistance signatures. Methods: Retrospectively, sCTC were isolated from 70 breast cancer (BC) patients using OncoRadar platform consisting affinity-based glass beads mediated by anti EpCAM antibody. CTCs were selectively captured and released without fixing and contamination of any leukocytes. Platform possess cleavable disulfide spacer at pH 8.0, between glass beads (diam. 2 mm, area 12.57 mm2) and a ligand. Sensitivity, specificity, positive predictive value (PPV), negative prediction value (NPV), and accuracy were evaluated using MCF-7 cells and CTCs. Assay to isolate CTC has been developed in 96 well plate, for downstream NGS-based comprehensive gene panel analysis using hybridization-based target enrichment. Interestingly, no mutations detected in blood samples (~20%) for CtDNA versus CTC DNA offered non-chip implicated mutations. Results:. Analytical validation using OncoRadar showed higher cell capture efficiency (99%) with spiked MCF-7 cells/ml of blood. Sensitivity and specificity values were 97% and 98% respect, while PPV was 99% and NPV was 7.5% with the accuracy parameter of 99%. Sensitivity, specificity, PPV, and NPV parameters when assessed for CTC isolation from 70 BC patients. NGS-based mutational analysis, sCTC showed the presence of PIK3CA, ESR1, and NF1 mutations, which were absent from paired ctDNA samples. Conclusion: Detection of ctDNA obscure mutations in paired sCTC samples suggests high sensitivity of sCTC-based genomic assays compared to current standard of practice. Heterogeneity scores amongst inter and intra CTCs with MATH scores offer new dimensions in genomics. Combination of ctDNA and sCTC DNA assay offers a comprehensive realistic genomic outcome for more significant therapy decisions, especially when guideline based options may exhausts. Citation Format: Gowhar Shafi, Atul Bharde, Ganesh Khutale, Saloni Andhari, Richa Deshpande, Bhagwat Jadhav, Sangeeta Prajapati, Moubeen Fauzul, Kanchan Hariramani, Madhura Basavalingegowda, Jayant Khandare. Capturing live single circulating tumor cells platform for comprehensive genomic profiling with paired ctDNA in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7507.

Towards genomic-Newborn Screening: Technical feasibility of Exome Sequencing starting from dried blood spots

Each year thousands of babies are born with rare genetic disorders not identified by current NBS panels, due to programs which are not yet optimal. Next-generation sequencing technologies have the potential to overcome many NBS drawbacks and provide large amounts of molecular data, broadening the number of diseases investigated. Here, we design and set up an NGS-based approach to evaluate the feasibility of NGS from dried blood spot starting from 34 DBSs.After assessing gDNA yield and integrity, libraries were performed using three target enrichment approaches, sequenced on NS500 platform, and analyzed on commercial platform. Specifically, we focus on virtual gene panels related to highly actionable neonatal/pediatric disorders.WES show that amount and quality of DBS-extracted gDNA are suitable for high-throughput sequencing. We obtain 500–1500 ng for each specimen, 1.7–1.8 260/280 wavelength, and DIN of 7 resulting DNA integrity, on par with traditional venous blood collection. A high read depth with 94.3% coverage uniformity is achieved for all samples.Data results on mean coverage are comparable among the different workflows tested and demonstrate that DBS from newborn collected at birth is a suitable material for the developing of gNBS programs.

Phylogenomics and morphology of Celmisiinae (Asteraceae: Astereae): Taxonomic and evolutionary implications

The tribe Astereae (Asteraceae) includes 36 subtribes and 252 genera, and is distributed worldwide in temperate and tropical regions. One of the subtribes, Celmisiinae Saldivia, has been recently circumscribed to include six genera and ca. 160 species, and is restricted to eastern Australia, New Zealand, and New Guinea. The species show an impressive range of growth habit, from small herbs and ericoid subshrubs to medium-sized trees. They live in a wide range of habitats and are often dominant in subalpine and alpine vegetation. Despite the well-supported circumscription of Celmisiinae, uncertainties have remained about their internal relationships and classification at genus and species levels. This study exploited recent advances in high-throughput sequencing to build a robust multi-gene phylogeny for the subtribe Celmisiinae. The target enrichment Angiosperms353 bait set and the hybpiper-nf and paragone-nf pipelines were used to retrieve, infer, and assemble orthologous loci from 75 taxa representing all the main putative clades within the subtribe. Because of the diploidised ploidy level in Celmisiinae, as well as missing data in the assemblies, uncertainty remains surrounding the inference of orthology detection. However, based on a variety of gene-family sets, coalescent and concatenation-based phylogenetic reconstructions recovered similar topologies. Paralogy and missing data in the gene-families caused some problems, but the estimated phylogenies were well-supported and well-resolved. The phylogenomic evidence supported Celmisiinae and three main clades: the Pleurophyllum clade (Pleurophyllum, Macrolearia and Damnamenia), mostly in the New Zealand Subantarctic Islands, Celmisia of mainland New Zealand and Australia, and Shawia (including ‘Olearia pro parte’ and Pachystegia) of New Zealand, Australia and New Guinea. The results presented here add to the accumulating support for the Angiosperms353 bait set as an efficient method for documenting plant diversity.

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a.

Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans-cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.