Detection Of Somatic Variants Research Articles

Abstract PO3-06-05: Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer

Abstract Background: Liquid biopsy provides a real-time assessment of metastatic breast cancer (MBC). Recently, the complementary prognostic value of tumor-derived extracellular vesicles (tdEVs) and circulating tumor cells (CTCs) has been reported. We have previously confirmed the strong prognostic significance of CTCs and tdEVs in inflammatory breast cancer (IBC). While previous studies have reported the association of CTCs with circulating tumor DNA (ctDNA) alterations in MBC, no evidence is available for tdEVs. This study aimed to analyze the association of tdEVs with ctDNA alterations, to investigate the molecular pathways underlying their presence. Moreover, we explored potential differences of this association in patients (pts) with IBC to provide a comprehensive liquid biopsy-based portrait of this aggressive subtype of BC. Methods: Blood samples were collected from 355 pts with MBC before starting a new line of therapy at Northwestern University (Chicago, IL) between 2016 and 2021 (NU16B06 trial). For CTCs and tdEVs analysis, 7.5 mL of blood was processed with the CellSearch® system. The ACCEPT software was applied to CellSearch® images to automatically enumerate CTCs and tdEVs. Positivity cutoff was ≥5 for CTCs, while tdEV cutoff points were < 20 (low), 20-79 (intermediate), and ≥80 (high). For ctDNA analysis, matched plasma samples (± 1 month) were analyzed with the Guardant360™ NGS platform for the detection of somatic single nucleotide variants (SNVs) and copy number variations (CNVs), which were classified into oncogenic pathways based on defined profiles generated on the Cancer Genome Atlas database (p53, PI3K, ER, RTK, RAS, RAF, WNT, MYC, cell cycle, notch). Associations between ctDNA pathways alterations and tdEVs were tested in the overall population by multinomial logistic regression and corrected for significant clinical characteristics. Differences in liquid biopsy features between IBC and non-IBC subgroups were analyzed through Fisher's exact test. Results: Of the 355 MBC pts, 210 (62%) had HR+ BC, 61 (17%) had HER2+ BC, and 68 (20%) had triple-negative BC. Eighty-three (23%) had a diagnosis of IBC and had a numerically lower tdEV count. Also, lower tdEVs were detected in HER2+ BC as compared with HR+. Significantly higher tdEVs were observed among pts with lobular histology, liver, and bone metastases. CTC count was significantly associated with tdEV number. A matched plasma sample for ctDNA analysis was available for 175 pts (62 IBC and 113 non-IBC). In the overall population, SNVs in ER pathway were associated with intermediated/high levels of tdEVs (p=0.004 and 0.008). A similar association was observed for CNVs in the cell cycle pathway (p=0.008 and 0.007). Moreover, associations with ≥80 tdEVs were observed for PI3K SNVs and CNVs (p=0.039 and 0.004, respectively), RTK CNVs (p=0.023), and MYC CNVs (p=0.001). In multivariable analysis, clinical characteristics associated with higher tdEVs were lobular histology (p=0.014 for 20-79 and < 0.001 for ≥80) and bone metastases (p=0.019 for 20-79 and 0.002 for ≥80). When considering clinical variables of interest, only ER SNVs and MYC CNVs were significantly associated with intermediate (p=0.031) and high (p=0.022) tdEV counts, respectively. While the association with ER SNVs and cell cycle CNVs was significant both in the IBC and non-IBC subgroups, others were specific for a certain subgroup: higher tdEVs were significantly associated with PI3K SNVs only in non-IBC pts whereas the significant association with CNVs in PI3K and MYC pathways was observed only in pts with IBC. Conclusion: Detection of tdEVs was associated with particular genomic profiles. These alterations seem to be different in IBC further underlying a different biology of this BC subtype. Additional studies are needed to explore how to integrate different liquid-biopsy based biomarkers in the management of pts with MBC. Citation Format: Eleonora Nicolò, Lorenzo Gerratana, Lorenzo Foffano, Laura Munoz Arcos, Mara Serena Serafini, Maroua Manai, Letizia Pontolillo, Nadia Bayou, Elisabetta Molteni, Amanda Kaylan Strickland, Youbin Zhang, Paolo D’Amico, Andrew Davis, Jeannine Donahue, Huiping liu, William Gradishar, Ami Shah, Giuseppe Curigliano, Carolina Reduzzi, Massimo Cristofanilli. Association of tumor-derived extracellular vesicles with circulating tumor DNA alterations in metastatic breast cancer patients: exploring differences in inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-06-05.

Comprehensive genomic profiling of pulmonary spindle cell carcinoma using tissue and plasma samples: insights from a real-world cohort analysis.

Pulmonary spindle cell carcinoma (PSCC) is a rare and aggressive non-small cell lung cancer (NSCLC) subtype with a dismal prognosis. The molecular characteristics of PSCC are largely unknown due to its rarity, which limits the diagnosis and treatment of this historically poorly characterized malignancy. We present comprehensive genomic profiling results of baseline tumor samples from 22 patients histologically diagnosed with PSCC, representing the largest cohort to date. Somatic genetic variant detection was compared between paired plasma samples and primary tumors from 13 patients within our cohort. The associations among genomic features, treatment, and prognosis were also analyzed in representative patient cases. TP53 (54.5%), TERT (36.4%), CDKN2A (27.3%), and MET (22.7%) were most frequently mutated. Notably, 81.8% of patients had actionable targets in their baseline tumors, including MET (22.7%), ERBB2 (13.6%), EGFR (9.1%), KRAS (9.1%), ALK (9.1%), and ROS1 (4.5%). The median tumor mutation burden (TMB) for PSCC tumors was 5.5 mutations per megabase (muts/Mb). TMB-high tumors (>10 muts/Mb) exhibited a significantly higher mutation frequency in genes such as KRAS, ARID2, FOXL2, and LRP1B, as well as within the DNA mismatch repair pathway. The detection rates for single nucleotide variants and structural variants were comparable between matched tumor and plasma samples, with 48.6% of genetic variants being mutually identified in both sample types. Additionally, a patient with a high mutation load and positive PD-L1 expression demonstrated a 7-month survival benefit from chemoimmunotherapy. Furthermore, a patient with an ALK-rearranged tumor achieved a remarkable 3-year progression-free survival following crizotinib treatment. Overall, our findings deepen the understanding of the complex genomic landscape of PSCC, revealing actionable targets amenable to tailored treatment of this poorly characterized malignancy.

Genetic Diagnosis of Retinoblastoma Using Aqueous Humour-Findings from an Extended Cohort.

The identification of somatic RB1 variation is crucial to confirm the heritability of retinoblastoma. We and others have previously shown that, when tumour DNA is unavailable, cell-free DNA (cfDNA) derived from aqueous humour (AH) can be used to identify somatic RB1 pathogenic variation. Here we report RB1 pathogenic variant detection, as well as cfDNA concentration in an extended cohort of 75 AH samples from 68 patients. We show cfDNA concentration is highly variable and significantly correlated with the collection point of the AH. Cell-free DNA concentrations above 5 pg/µL enabled the detection of 93% of known or expected RB1 pathogenic variants. In AH samples collected during intravitreal chemotherapy treatment (Tx), the yield of cfDNA above 5 pg/µL and subsequent variant detection was low (≤46%). However, AH collected by an anterior chamber tap after one to three cycles of primary chemotherapy (Dx1+) enabled the detection of 75% of expected pathogenic variants. Further limiting our analysis to Dx1+ samples taken after ≤2 cycles (Dx ≤ 2) provided measurable levels of cfDNA in all cases, and a subsequent variant detection rate of 95%. Early AH sampling is therefore likely to be important in maximising cfDNA concentration and the subsequent detection of somatic RB1 pathogenic variants in retinoblastoma patients undergoing conservative treatment.

Abstract 326: Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method

Abstract In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. It is difficult to obtain libraries with sufficient coverage and the sequencing artifacts arising from damaged DNA bases confound somatic variant detection. Additionally, many laboratories process FFPE tumor samples alongside matched, high quality, normal DNA and many library prep workflows are not readily compatible with both sample types. We developed a novel NGS library prep method compatible with both high quality and very low quality FFPE DNA samples, employing a novel enzymatic DNA repair mix, enzymatic fragmentation mix, and PCR master mix. To validate this workflow on real patient sample sets, we obtained DNA extracted from matched tumor and normal tissue of various tissue types preserved by both fresh frozen and FFPE methods. The FFPE DNA samples ranged in quality from DNA integrity number (DIN) 1.5 to 6.8, including samples stored for nearly 3 decades. We prepared libraries using this method compared against other library prep workflows and sequenced them by WGS and target capture. We assessed the performance of these workflows by library yield, library quality metrics, depth and evenness of coverage, and somatic variant calling with fresh frozen-extracted DNA providing the gold standard for library quality and mutation content. This new enzymatic fragmentation-based library prep workflow not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations in FFPE DNA samples but also improved the library yield, library quality metrics (including mapping, chimeras, and properly-paired reads), library complexity, coverage depth and uniformity, and hybrid capture library quality metrics. Comparing the variant calls from matched FFPE and frozen tissues revealed an improved sensitivity and accuracy of variant calling using this library prep method compared to mechanical shearing and other enzymatic fragmentation library prep approaches. This new suite of enzyme mixes allows even highly damaged FFPE samples to achieve high quality libraries with a greater sensitivity for somatic variant identification. The workflow is robust and flexible, compatible with both FFPE DNA and matched high quality DNA samples as well as automation-friendly for convenience in sample processing. Citation Format: Margaret R. Heider, Jian Sun, Brittany Sexton, Bradley W. Langhorst, Laura Blum, Pingfang Liu. Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method [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 326.

Abstract 3886: Analysis of clonal heterogeneity within paired primary and metastatic tumor samples of patients with solid tumors and implications for neoantigen-based personalized cancer vaccines

Abstract Background: Neoantigen-based personalized cancer vaccines carry significant promise in treating solid malignancies. However, there are uncertainties regarding the choice between the primary or the metastatic tumor for neoantigen prediction in individual patients. Here, we conducted a thorough examination of somatic variations in 676 patients who had paired primary and metastatic solid tumors. Methods: Patients were enrolled in the Total Cancer Care protocol (NCT03977402) to which patients provided an IRB-approved written informed consent within the Oncology Research Information Exchange Network (ORIEN). Whole-exome sequencing of 756 primary and metastatic tumor pairs was performed (N = 676 patients). These included Genitourinary (n=83), Gynecological (n=97), Gastrointestinal (n=213), Thoracic (n=33), Cutaneous (n=24), Breast (n=108), Endometrial (n=49), Sarcoma (n=35), Head-and-Neck (n=106) and others (n=8). The data was analyzed through the ORIEN AVATAR Molecular Analysis Pipeline for somatic mutation variant detection and variant annotation. In this analysis, we focused on somatic events that result in an in-frame alteration (such as missense, in-frame deletion and in-frame insertion) and out-of-frame protein-altering mutations (such as frameshifts, de novo start, out-of-frame, and nonstop gain). Clonal population structure was determined based on pyclone-vi. Results: For in-frame events, bladder cancer, melanoma, and gynecological cancers shared close to 75% of the mutations between paired primary and metastatic cases. In contrast, sarcoma and thyroid cancer had a low overlap (~ 25%) of variants. For out-of-frame events, these events tend to have a lower proportion of shared somatic variants between primary and metastasis than in-frame variants. Oncogenic drivers (e.g., BRAF V600E, KRAS G12A, and TP53 loss-of-function) were highly likely to be present in both paired primary and metastatic tumors. Next, we performed additional analysis on evolutionary selection of protein-coding variants via dN/dS calculation. We found no significant global shift in dN/dS ratio between paired primary and metastatic tumors across malignancies. However, we found increased selection of protein-coding variants in brain and liver metastatic sites, which correlated with increased homologous recombination deficiency within these sites. Conclusions: Our analysis demonstrates genetic variations that exist when comparing paired primary and metastatic tumors that appear to vary by histology. Variants are potentially undergoing negative selection supported by the preferential loss of out-of-frame events in metastatic tumors and positive selection in specific metastatic sites. Overall, understanding the clonal structure will be key to neoantigen prediction for effective neoantigen-based vaccines. Citation Format: Alyssa Obermayer, Timothy Shaw, Darwin Chang, Joshua Davis, Jamie K. Teer, Xiaoqing Yu, Xuefeng Wang, Dale Hedges, Aik Choon Tan, Robert Rounbehler, Abdul Rafeh Naqash, Margaret Gatti-Mays, Aakrosh Ratan, Martin McCarter, Howard Colman, Igor Puzanov, Susanne Arnold, Michelle Churchman, Patrick Hwu, William Dalton, George Weiner, Jose Conejo-Garcia, Paulo C. Rodriguez, Bodour Salhia, Ahmad A. Tarhini. Analysis of clonal heterogeneity within paired primary and metastatic tumor samples of patients with solid tumors and implications for neoantigen-based personalized cancer vaccines [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 3886.

Abstract 1771: NYU Langone Genome PACT - Genome Profiling of Actionable Cancer Targets (LG-PACT) for clinical patient molecular diagnostics and treatment

Abstract Next-generation sequencing (NGS) for the detection of somatic variants has become the tool of choice in a variety of molecular oncology fields and in the clinic. Its use ranges from sequencing entire tumor genomes and transcriptomes to targeted clinical diagnostic gene panels. The NYU Langone Genome PACT assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients to detect gene alterations in a 607-gene panel. Indications for testing are cancer (solid tumors or hematological malignancies) where a mutational profile from multiple genes would be informative for disease stratification, prognosis, or treatment options including targeted therapies and eligibility for clinical trials. The test is intended to provide information on somatic mutations including point mutations, copy number aberrations, and small insertions/deletions (indels) for diagnostic and treatment decisions. LG-PACT is a United States Food and Drug Administration (FDA) approved diagnostic test. The clinical interpretation of sequencing data of molecular tumor markers from NGS encompasses automated variant calling tools with human interpretation. The final mostly manual review of data is intensive, involving highly trained scientists, encompassing literature review, interpretation and tier classification by pathologists, who then provide a complete molecular diagnostic report to the treating oncologists. Since January 2022 to present (October 2023) we have provided clinical genomic reports for 1029 oncological cases from 124 different cancers and their subtypes, including Brain (489 cases incl. meningioma, glioma and glioblastoma), Gastrointestinal (123), Lung (114), among others. We first present the technical challenges of validating an NGS oncological diagnostic targeted assay for appropriate clinical grade accuracy and sensitivity acceptable for patient care. We show how copy number alterations provide a more comprehensive description of the tumors genomic profile. We then outline the actionability of targeted panel sequencing for our current patient cohort. Where analysis of variant detection has led to 47% (490) of our clinical tumor samples containing known tier 1 therapeutic variants. We conclude with presenting case studies that identify both the clinical utility and informatic challenges of variant calling specific to gene panel sequencing e.g., i) the capture of potential targetable rare and novel indels and multivariant mutations in exons 19 and 20 of EGFR, and ii) a unique KIT tandem duplication event in a gastrointestinal stromal tumor (GIST). We demonstrate the value in precision oncology for multiple cancer types and how the capture of unique variants can provide better targeted treatment options to cancer patients. Citation Format: Varshini Vasudevaraja, Yiying Yang, Jonathan Serrano, Kazimierz Wrzeszczynski, Matija Snuderl. NYU Langone Genome PACT - Genome Profiling of Actionable Cancer Targets (LG-PACT) for clinical patient molecular diagnostics and treatment [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 1771.

Abstract 4926: Advancements in somatic variant calling from UG100 whole genome and whole exome sequencing data

Abstract Somatic variant calling involves the identification of genomic alterations that occur in somatic cells, requiring deep coverage to enable high sensitivity for low-frequency variants. Characterizing somatic variants across the entire genome therefore benefits from novel cost-efficient sequencing platforms, such as UG100. Here, we present optimization of variant calling tools for short and structural variants on WGS and WES data from UG100. For calling short variants, we optimized DeepVariant (DV) for somatic calling using data from matched tumor-normal sample pairs, improving both variant calling accuracy and pipeline running time (up to 10-fold). We defined the task of somatic variant calling as deciding if the pileup image containing reads from the tumor and normal samples represents a true somatic variant (vs a germline variant or artifact). The challenge of robust variant calling using deep learning models is exacerbated in somatic calling, where sequencing depth and coverage variability are typically high. Our optimized DV overcomes these challenges by several data sampling strategies. First, allele-frequency preserving down-sampling reduces randomness of read sub-sampling in high coverage regions. Second, alternative allele prioritization samples alt-allele supporting reads first allowing to call variants at very high coverage loci without sacrificing sensitivity and computational efficiency. Finally, a Panel-of-Normals based on targeted WES data provides an additional improvement of precision for this assay type. We used these strategies to train two models, one for tumor characterization using WGS (T/N coverage: 40x-150x/40x-100x), and one for deep WES (T/N coverage: >500x/>120x). We called variants on simulated tumors using the WGS model. For VAF>10% the model showed SNV recall >98% and indel recall >95% with false-positive rate of 0.2/Mb. For VAF range of 5-10%, indel recall was 67% and SNV recall was 86%. To demonstrate the utility of our somatic variant calling, we applied the models to call somatic variants from well characterized cancer cell lines: COLO829, HCC1395 and HCC1143. Results showed F1>90% for variants with VAF>10%. The WES model was used to reliably call variants at VAF>5% on simulated tumors with average SNV recall of 99% with precision >99% and indel recall >86% with precision >94%. To analyze structural and copy-number variations, we optimized the assembly engine of GRIDSS to enable fast calling of structural variations and demonstrate that Control-FREEC can be used to call copy number variants. SV calling on COLO829/COLO829BL achieved sensitivity >95%. In conclusion, our research highlights the utility of UG100 within the field of oncology, demonstrating its capacity for comprehensive and precise somatic variant detection, both on WGS and WES data. Citation Format: Doron Shem-Tov, Maya Levy, Gil Hornung, Ilya Soifer, Hila Benjamin, Ariel Jaimovich, Adam Blattler, William Brandler, Robert Sugar, Isaac Kinde, Omer Barad, Doron Lipson. Advancements in somatic variant calling from UG100 whole genome and whole exome sequencing data [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 4926.

Abstract 2934: Somatic variant workflow with HiFi sequencing provides new insights in highly challenging cancer cases

Abstract Tumor evolution is a highly heterogeneous process where multiple oncogenic pathways can lead to host defense evasion. Advancements in genome sequencing are allowing us to better understand these processes and their impact. However, critical genomic variations that could be pivotal to the development of tumors may have remained undetected due to technological constraints of short-read sequencing. PacBio HiFi sequencing generates accurate (>99.9%) long reads (>15 kb) with native 5-methylcytosine information that can comprehensively delineate many variations, including ones which were previously inaccessible with short-read sequencing. Unlike standard whole-genome analysis, cancer variant calling must account for somatic variation, where the assumptions of a diploid genome are violated. We have developed and optimized a bioinformatics workflow for somatic variant detection with HiFi sequence data. To validate this pipeline, we sequenced two well-studied cancer cell lines (COLO829, HCC1395) to 60-fold depth and their matched normal lymphoblast cells (40-fold to 60-fold depth) and called all classes of variations (SNVs, INDELS, and structural variants). Next, we compared our calls against the Valle-Inclan (structural variants) and SEQC2 (small variants: SNV/INDEL) datasets. We achieved 92% F1 score for small variants across all variant allele frequencies (VAF) in the HCC1395 experiment. We observed a maintained accuracy (F1=91%) even at lower variant frequencies (10-20), highlighting the ability of HiFi sequencing to detect subclonal mutations. The workflow calls structural variants with high sensitivity, capturing 57/62 (92%) of all previously validated SVs in COLO829 at all VAF. Lastly, we applied our pipeline to five diverse tumor samples that exhibit complex oncogenic phenotypes including homologous recombination deficiency (HRD) and high genomic instability, e.g. unresolved rearrangement breakpoints in repetitive regions and unknown oncogenic drivers when prior sequencing efforts did not provide full clarity as to the molecular drivers of tumorigenesis. Notably, we were able to identify and phase 5mC methylation of promoters together with large-scale structural variants in cancer genes involved in HRD. Taken together, we demonstrated the high accuracy of HiFi reads in resolving somatic variants and its potential to provide novel insights in complex cancer samples. The ability for long reads to resolve complex genomics aberrations and epigenetic markers that underly the tumorigenesis process will become indispensable in the realm of precision oncology. Citation Format: Khi Pin Chua, Ian McLaughlin, Oliver Hofmann, Kym Pham Stewart, Primo Baybayan, Jonathan Bibliowicz, Sean Grimmond, Zev Kronenberg, Michael A. Eberle. Somatic variant workflow with HiFi sequencing provides new insights in highly challenging cancer cases [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 2934.

Improving somatic exome sequencing performance by biological replicates

BackgroundNext-generation sequencing (NGS) technologies offer fast and inexpensive identification of DNA sequences. Somatic sequencing is among the primary applications of NGS, where acquired (non-inherited) variants are based on comparing diseased and healthy tissues from the same individual. Somatic mutations in genetic diseases such as cancer are tightly associated with genomic instability. Genomic instability increases heterogenity, complicating sequencing efforts further, a task already challenged by the presence of short reads and repetitions in human DNA. This leads to low concordance among studies and limits reproducibility. This limitation is a significant problem since identified mutations in somatic sequencing are major biomarkers for diagnosis and the primary input of targeted therapies. Benchmarking studies were conducted to assess the error rates and increase reproducibility. Unfortunately, the number of somatic benchmarking sets is very limited due to difficulties in validating true somatic variants. Moreover, most NGS benchmarking studies are based on relatively simpler germline (inherited) sequencing. Recently, a comprehensive somatic sequencing benchmarking set was published by Sequencing Quality Control Phase 2 (SEQC2). We chose this dataset for our experiments because it is a well-validated, cancer-focused dataset that includes many tumor/normal biological replicates. Our study has two primary goals. First goal is to determine how replicate-based consensus approaches can improve the accuracy of somatic variant detection systems. Second goal is to develop highly predictive machine learning (ML) models by employing replicate-based consensus variants as labels during the training phase.ResultsEnsemble approaches that combine alternative algorithms are relatively common; here, as an alternative, we study the performance enhancement potential of biological replicates. We first developed replicate-based consensus approaches that utilize the biological replicates available in this study to improve variant calling performance. Subsequently, we trained ML models using these biological replicates and achieved performance comparable to optimal ML models, those trained using high-confidence variants identified in advance.ConclusionsOur replicate-based consensus approach can be used to improve variant calling performance and develop efficient ML models. Given the relative ease of obtaining biological replicates, this strategy allows for the development of efficient ML models tailored to specific datasets or scenarios.

Abstract 2291: Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation

Abstract Background: Current genomic and epigenomic profiling of cancer tissue DNA or cfDNA (cell-free DNA) in liquid biopsy relies upon separate, time- and sample-consuming technologies for somatic variant detection or methylation analysis. Here we describe workflow and performance of the Agilent Bravo automated liquid handling platform with the Agilent Avida targeted enrichment solution for next generation sequencing of somatic variants and methylation profiling. This solution can effectively analyze low-input tumor DNA or cfDNA samples. The Avida Duo workflow enables highly sensitive detection of single nucleotide variant (SNV), insertions and deletions (INDEL), copy number variation (CNV) and DNA methylation profiles from a single sample, without any sample splitting. Methods and Results: Panels, reagents, and automated workflows for Avida DNA, Avida Methyl, and Avida Duo Methyl (combined DNA & methylation) kits were developed to accommodate up to 96 samples on the Bravo NGS workstation. The automated solution supports independent single-day workflows for somatic variants or methylation sample preparation. The Avida Duo analysis combines both workflows without sample splitting, streamlining the process and reducing sample consumption. Leveraging a focused cancer hotspot panel, we demonstrate excellent reproducibility and low allele frequency (SNV at sub 1%) variant detection in cfDNA samples and reference standards with as little as 10ng DNA input. We exhibit similar performance with a larger ~300 kb target region tumor profiling panel. Finally, we demonstrate DNA methylation detection in the standalone or combined (“Duo”) workflows across differentially methylated regions (DMRs) with a panel of ~3400 targets. Conclusions: Automation of the Avida targeted enrichment solution with the Bravo NGS workstation enables a sensitive, high-throughput, end-to-end analysis for detection of genomic alterations and DNA methylation changes from a single low-input cfDNA or tumor DNA sample. Citation Format: Ashraf Wahba, Tony Ho, Sarah Johns, Aswati Aravind, Heng Wang, Neelima V. Mehendale, Gilbert P. Amparo, Khine Win, Manuel Gomez, Grace Zhao, Douglas N. Roberts. Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation [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 2291.

Integrating Optical Genome Mapping and Whole Genome Sequencing in Somatic Structural Variant Detection.

Structural variants drive tumorigenesis by disrupting normal gene function through insertions, inversions, translocations, and copy number changes, including deletions and duplications. Detecting structural variants is crucial for revealing their roles in tumor development, clinical outcomes, and personalized therapy. Presently, most studies rely on short-read data from next-generation sequencing that aligns back to a reference genome to determine if and, if so, where a structural variant occurs. However, structural variant discovery by short-read sequencing is challenging, primarily because of the difficulty in mapping regions of repetitive sequences. Optical genome mapping (OGM) is a recent technology used for imaging and assembling long DNA strands to detect structural variations. To capture the structural variant landscape more thoroughly in the human genome, we developed an integrated pipeline that combines Bionano OGM and Illumina whole-genome sequencing and applied it to samples from 29 pediatric B-ALL patients. The addition of OGM allowed us to identify 511 deletions, 506 insertions, 93 duplications/gains, and 145 translocations that were otherwise missed in the short-read data. Moreover, we identified several novel gene fusions, the expression of which was confirmed by RNA sequencing. Our results highlight the benefit of integrating OGM and short-read detection methods to obtain a comprehensive analysis of genetic variation that can aid in clinical diagnosis, provide new therapeutic targets, and improve personalized medicine in cancers driven by structural variation.

Analytical validation and clinical utilization of K-4CARE™: a comprehensive genomic profiling assay with personalized MRD detection.

Background: Biomarker testing has gradually become standard of care in precision oncology to help physicians select optimal treatment for patients. Compared to single-gene or small gene panel testing, comprehensive genomic profiling (CGP) has emerged as a more time- and tissue-efficient method. This study demonstrated in-depth analytical validation of K-4CARE, a CGP assay that integrates circulating tumor DNA (ctDNA) tracking for residual cancer surveillance. Methods: The assay utilized a panel of 473 cancer-relevant genes with a total length of 1.7Mb. Reference standards were used to evaluate limit of detection (LOD), concordance, sensitivity, specificity and precision of the assay to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), gene amplification and fusion, microsatellite instability (MSI) and tumor mutational burden (TMB). The assay was then benchmarked against orthogonal methods using 155 clinical samples from 10 cancer types. In selected cancers, top tumor-derived somatic mutations, as ranked by our proprietary algorithm, were used to detect ctDNA in the plasma. Results: For detection of somatic SNVs and Indels, gene fusion and amplification, the assay had sensitivity of >99%, 94% and >99% respectively, and specificity of >99%. Detection of germline variants also achieved sensitivity and specificity of >99%. For TMB measurement, the correlation coefficient between whole-exome sequencing and our targeted panel was 97%. MSI analysis when benchmarked against polymerase chain reaction method showed sensitivity of 94% and specificity of >99%. The concordance between our assay and the TruSight Oncology 500 assay for detection of somatic variants, TMB and MSI measurement was 100%, 89%, and 98% respectively. When CGP-informed mutations were used to personalize ctDNA tracking, the detection rate of ctDNA in liquid biopsy was 79%, and clinical utility in cancer surveillance was demonstrated in 2 case studies. Conclusion: K-4CARE™ assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapy and immunotherapy. Integration of ctDNA tracking helps clinicians to further monitor treatment response and ultimately provide well-rounded care to cancer patients.

Cell Tree Rings: the structure of somatic evolution as a human aging timer

Biological age is typically estimated using biomarkers whose states have been observed to correlate with chronological age. A persistent limitation of such aging clocks is that it is difficult to establish how the biomarker states are related to the mechanisms of aging. Somatic mutations could potentially form the basis for a more fundamental aging clock since the mutations are both markers and drivers of aging and have a natural timescale. Cell lineage trees inferred from these mutations reflect the somatic evolutionary process, and thus, it has been conjectured, the aging status of the body. Such a timer has been impractical thus far, however, because detection of somatic variants in single cells presents a significant technological challenge. Here, we show that somatic mutations detected using single-cell RNA sequencing (scRNA-seq) from thousands of cells can be used to construct a cell lineage tree whose structure correlates with chronological age. De novo single-nucleotide variants (SNVs) are detected in human peripheral blood mononuclear cells using a modified protocol. A default model based on penalized multiple regression of chronological age on 31 metrics characterizing the phylogenetic tree gives a Pearson correlation of 0.81 and a median absolute error of ~4 years between predicted and chronological ages. Testing of the model on a public scRNA-seq dataset yields a Pearson correlation of 0.85. In addition, cell tree age predictions are found to be better predictors of certain clinical biomarkers than chronological age alone, for instance glucose, albumin levels, and leukocyte count. The geometry of the cell lineage tree records the structure of somatic evolution in the individual and represents a new modality of aging timer. In addition to providing a numerical estimate of “cell tree age,” it unveils a temporal history of the aging process, revealing how clonal structure evolves over life span. Cell Tree Rings complements existing aging clocks and may help reduce the current uncertainty in the assessment of geroprotective trials.

Exploring the regulatory interactions between mutated genes and homeobox genes in the head and neck cancer progression

ObjectiveUnderstanding the regulatory role of homeobox (HOX) and mutated genes in the progression of head and neck cancers is essential, although their interaction remains elusive. This study aims to decipher the critical regulation of mutation driven effects on homeobox genes to enhance our understanding of head and neck cancer progression. MethodsGenomic mutation data from The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma were analyzed using VarScan2 for somatic variant detection. Mutational clustering, driver mutation identification, and cancer signaling pathway analysis were performed using the OncodriveCLUST method. Harmonizome datasets were retrieved to identify critical cancer driver genes affecting HOX genes. The effects of HPV infection on HOX and mutated genes were assessed using the oncoviral database. Altered pathway activity due to the effects of cancer drivers on HOX genes was analyzed with Gene Set Cancer Analysis. Functional enrichment analysis of gene ontology biological processes and molecular functions was conducted using the ClusterProfiler R package. ResultsSignificant alterations in HOX genes were observed in head and neck cancer cohorts with mutated TP53, FAT1, and CDKN2A. HOX genes were identified as functionally downstream targets of TP53, signifying transcriptionally mediated regulation. The interaction between HOX genes and mutated TP53, FAT1, and CDKN2A dysregulated the epithelial-to-mesenchymal transition, cell cycle, and apoptosis pathways in head and neck cancer progression. ConclusionThe interplay between cancer driver genes and HOX genes is pivotal in regulating the oncogenic processes underlying the pathogenesis of head and neck squamous cell carcinoma

Comment on: Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

Comment on: Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

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Comment on: Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome: reply.

Usefulness of Peripheral Blood Samples in Diagnosis and Prognosis Assessment of Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia Low Risk Patients Using Next Generation Sequencing

Introduction: myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Morphologic bone marrow (BM) examination, cytogenetic analysis and molecular techniques are indispensable for accurate diagnosis and disease classification. BM constitutes the most suitable sample for molecular studies, however, peripheral blood (PB) can be obtained by minimally invasive venipuncture and would be ideal for comprehensive sequential monitoring of MDS over time. Aim: to assess whether targeted deep sequencing (TDS) allows the detection of somatic variants from diagnosis and follow-up PB samples and if the molecular profiles are comparable to those from BM samples. Methods: preliminary results include the study of 32 low risk patients (28 MDS and 4 chronic myelomonocytic leukemia, CMML). At least one sample, either BM or PB, was obtained at the moment of diagnosis and whenever possible, paired samples obtained the same day were collected. Over the course of the disease only PB samples were obtained. Libraries were prepared for all available samples using DNA from whole BM or whole PB using a custom hybridization-probe based panel (KAPA HyperCap, Roche), including selected exons of 50 myeloid-related genes. TDS was performed on Illumina MiSeq instrument at a mean coverage of 1000x. Data were analyzed using local bioinformatic pipeline. Only those variants with ≥100x locus coverage and ≥25 reads for the alternative allele (quality criteria) were considered for downstream analysis. Variants were filtered according to variant type, location, read depth (>100x) and population frequency (<1%). Results: patients´ characteristics are described in table 1. The combination of cytogenetics and TDS identified genetic alterations in 29/32 patients (91%). TDS allowed the identification of somatic variants in 27/32 patients (85%), being TET2 (n=23, 29%), SF3B1 (n=12, 15%) and ASXL1 (n=8, 10%) the most frequently mutated genes. Considering molecular information, restratification of IPSS-R to IPSS-M risk groups of MDS patients (n=28) was performed (Table 1). As previously described (Sauta et al, J Clin Oncol, 2023), we performed a five-to-five comparison of IPSS-R and IPSS-M patients' distribution (merging moderate low and moderate high to moderate in IPSS-M). This resulted in the restratification of 36% of MDS patients (10/28) of which 6% (n=2) were downstaged and 25% (n=8) were upstaged. Suitability of PB samples for NGS analysis was evaluated in 21 patients in which paired BM and PB samples were obtained at diagnosis. Overall, 65 variants were found, and 63 of them were detectable in both BM and PB samples. Despite the other two variants were also detected in PB samples at low variant allele frequency (VAF) values ( TET2 and NRAS, VAF=1,5% and 0,99%, respectively), they strictly did not meet our quality criteria (≥25 reads for the alternative allele). Nevertheless, a strong correlation was identified between VAF values for the individual variants detected in BM vs. PB (Figure 1). As part of this project, molecular profile during patients' follow-up is being studied using PB samples. To date, at least one follow-up sample of each patient was studied. The mean time between diagnosis and the first follow-up sample was 28 months (range 6-84 months). After this time, and despite molecular profiles are heterogeneous, variations could be detected in up to 72% of patients (23/32) using PB samples. The most commonly detected variation was an increasing in VAF values (9/23; defined by a difference >5% in relation to the VAF detected in the previous sample), followed by a new variant detection + increasing in VAF values (7/23), new variant detection only (6/23) and a single case where simultaneous decreasing VAF and disappearance of subclones was detected (single patient receiving treatment with hypomethylating agent). Conclusion: molecular testing has become increasingly important for accurate diagnosis and risk assessment in MDS and CMML patients, specially with the new classifications and prognostic scoring system. Here we demonstrate that the molecular analysis from PB cells constitutes an appropriate alternative for the detection and quantification of somatic variants in MDS patients at diagnosis and during disease course. Funding: Bristol Myers Squibb (PI-13279-IIT), FEHH and 2021 SGR 00560 (GRC).

Early Identification of Refractory/Relapsed Diffuse Large B Cell Lymphoma with Serial Ctdna Sampling

Background: Diffuse Large B Cell Lymphoma (DLBCL) is an aggressive lymphoma that is curable in 60% of patients with chemoimmunotherapy. Outcomes are poor for those experiencing relapsed or refractory disease (rrDLBCL), particularly for patients with primary refractory disease (REFR, < 9 months from diagnosis) and early relapse (ER, 9 months to 2 years from diagnosis). Some of these patients would be candidates for chimeric antigen receptor T cell therapy (CART) in second line, where outcomes are superior when the disease burden is low. Therefore, exploring strategies to identify these high-risk patients early is important. Plasma circulating tumor DNA has been shown to be prognostic in various DLBCL cohorts. Method: We developed a custom panel of 170 genes to identify early treatment failure in a cohort of 171 patients that had profiling performed on 323 plasma samples. Plasma samples were taken serially starting at diagnosis and as patients progressed through frontline treatment. All plasma samples underwent DNA extraction, library preparation and subsequent sequencing using a panel of DLBCL related genes at high read depth (~1000x). Single nucleotide variant (SNV) calling was carried out using a custom pipeline including paired normal DNA for improved somatic variant detection. ctDNA fraction was estimated based on the highest variant allele fraction detected, using a loss of heterozygosity somatic model. Our ctDNA analysis focused on samples at diagnosis, cycle 2 of therapy, and end of treatment in order to identify early determinants of refractory disease. Changes in ctDNA levels between time points were represented as log2 ratio of ctDNA fraction. Results: rrDLBCL cases were separated into 3 categories based on the time between diagnosis and progressive disease (PD): 47 REFR, 34 ER, and 31 late relapse (LR, >24 months). The remaining patients were disease free after frontline therapy for over 24 months (CR, n=59), resulting in a cohort enriched for rrDLBCL (65% of cases). The average international prognostic index (IPI) of each group at diagnosis was REFR=3.38, ER=2.87, LR=2.81, and CR=2.41. Cell of Origin, as determined by Hans algorithm, showed a higher number of non-GCB samples in ER and LR groups (53% and 60%, respectively) while REFR and CR displayed increased GCB cases (63% and 66%, respectively). The REFR patients were significantly more likely to be of a 4/5 IPI score at diagnosis than other groups (p=0.0203). Progression-free survival (PFS2) at relapse therapy for REFR (median=0.29 years) was shorter when compared to both ER (p=0.0104, median=0.44 years) and LR (p=0.0012, median=0.80 years). Using the diagnostic plasma sample, there was no significant difference in ctDNA fraction in any of the three groups. When the sample with the highest ctDNA fraction was compared between patients, REFR had significantly higher levels than LR and CR, consistent with a higher overall tumor burden (p=0.001 and 0.026, respectively). There was a trend towards higher ctDNA fraction at the end of treatment in both REFR and ER groups. As ctDNA dynamics are known to be informative of molecular response, we compared patients using the change in ctDNA fraction at cycle 2 of therapy (log2 ratio). This value was significantly different in REFR patients (p=0.0073 vs ER), consistent with a lower rate of molecular response to treatment. Conclusions: While refractory and later relapsed DLBCL have similar ctDNA features at diagnosis, we find they have distinct ctDNA dynamics during treatment. REFR had higher maximal ctDNA levels across time points and both REFR and ER patients exhibited higher levels of ctDNA at end of treatment. Moreover, patients with minimal change in ctDNA levels at cycle 2 of therapy are at a high risk of treatment failure and refractory disease. Further exploration of the specific mutational patterns is ongoing. These results could enable the earlier identification of rrDLBCL and facilitate the prioritization of approaches for therapeutic intervention in rrDLBCL.

Validation of targeted next-generation sequencing of cell-free DNA from archival cerebrospinal fluid specimens for the detection of somatic variants in cancer involving the leptomeninges: Cytopathologic and radiographic correlation.

Leptomeningeal metastases occur across multiple solid and lymphoid cancers, and patients typically undergo cytopathologic assessment of cerebrospinal fluid (CSF) in this setting. For patients diagnosed with metastatic cancer, the detection of actionable somatic mutations in CSF can provide clinically valuable information for treatment without the need for additional tissue collection. The authors validated a targeted next-generation sequencing assay for the detection of somatic variants in cancer (OncoPanel) on cell-free DNA (cfDNA) isolated from archival CSF specimens in a cohort of 25 patients who had undergone molecular testing of a prior tumor specimen. CSF storage time and volume had no impact on cfDNA concentration or mean target coverage of the assay. Previously identified somatic variants in CSF cfDNA were detected in 88%, 50%, and 27% of specimens diagnosed cytologically as positive, suspicious/atypical, and negative for malignancy, respectively. Somatic variants were identified in 81% of CSF specimens from patients who had leptomeningeal enhancement on magnetic resonance imaging compared with 31% from patients without such enhancement. These data highlight the stability of cfDNA in CSF, which allows for cytopathologic evaluation before triage for next-generation sequencing assays. For a subset of cases in which clinical suspicion is high but cytologic or radiographic studies are inconclusive, the detection of pathogenic somatic variants in CSF cfDNA may aid in the diagnosis of leptomeningeal metastases.

B-249 Usefulness of Simultaneous Detection of Germline and Somatic Variants From Different Specimen Types by Next-generation Sequencing

Abstract Background Genetic variants detection by next-generation sequencing (NGS) from tumor specimen only is considered limited in diagnosis due to difficulties in discriminating somatic variants from germline ones. It will be even more helpful in diagnosing patients with cancer if we perform NGS analysis from specimen of both tumor and blood. This study shows the usefulness of simultaneous detection of germline and somatic variants from different specimen types by NGS. Methods NGS panel assay designed for solid tumor composed of 80 genes was conducted for detection of both somatic and germline variants. DNA samples extracted from both buffy coat of whole blood and formalin-fixed paraffin-embedded (FFPE) specimen from 4 patients with renal cancer and 3 patients with ovarian cancer were used in assay. We compared results from both specimen and finalized somatic and germline variants detected. Results In 4 patients specimen with renal cell carcinoma, we were able to rule out germline variants from somatic variants by analysis of NGS assay from both specimen types. Genes ruled out for germline variants of uncertain significance (tier 3) or higher levels are ARID1B: c.1031C>T, p.A344V, ERBB2: c.3430G>C, p.D1144H, BRCA2: c.3220A>T, p.S1074C, POLE: c.2974G>A, p.A992T, EP300: c.1666A>G, p.M556V, EP300: c.2539C>T, p.P847S, ERBB3: c.1115C>G, p.P372R, BAP1: c.1124A>T, p.E375V. In 3 patients with ovarian cancer, genes ruled out are EGFR: c.739G>C, p.D247H, MET: c.4172A>T, p.D1391V, PBRM1: c.2678A>G, p.Y893C, SETD2: c.1208G>A, p.S403N, AKT1: c.1373T>C, p.M458T, FGFR3: c.160G>A, p.G54R, KMT2C: c.9530G>A, p.R3177H, SMARCB1: c.1116G>A, p.T372T. All variants confirmed are tier 3 level of clinical significance. Conclusion According to the results of this study, we were able to exclude germline variants form somatic ones in NGS assay reports for cancer patients. This study shows the usefulness of simultaneous detection of germline and somatic variants from different specimen types by NGS.