Genomic Profiling Of Solid Tumors Research Articles

Clinical validation of Northstar Select, a novel liquid biopsy assay for comprehensive genomic profiling of solid tumors.

3072 Background: Comprehensive Genomic Profiling (CGP) liquid biopsy tests are vital tools for oncologists that enable the non-invasive detection of diverse somatic mutations, many of which correspond to targeted therapies, in circulating tumor DNA (ctDNA). However, these tests have less utility when ctDNA signal is low, which is common even in late-stage cancer patients. BillionToOne’s Northstar (NS) Select assay is a novel CGP test that seeks to leverage proprietary technology and novel calling algorithms to address this limitation by achieving higher sensitivity. Methods: Clinical samples for 182 unique patients with stage III or IV solid tumors were obtained via a prospective validation study assessing head-to-head concordance with widely adopted, commercially available ‘comparator’ liquid biopsy CGP assays. The study cohort was recruited from 1 large hospital center and 6 community clinics across the US and was diverse in patient demographics and clinical presentations. Over 15 different cancer types were represented in the cohort, with the majority of patients having either Lung, Breast, Colorectal, or Prostate cancer, all of which are commonly tested using liquid biopsies in the US. Pathogenic (including likely pathogenic) variants called by NS Select and/or the comparator assay were analyzed for concordance. For a subset of samples (N=28), white blood cell-derived genomic DNA was also processed to identify which variants were detected due to clonal hematopoiesis of indeterminate potential (CHIP). Results: In the head-to-head comparison, a large number of variants (N=380) were concordant between the two assays. In addition, NS Select detected 43% (N=147) more pathogenic variants than comparators when controlling for matched coverage regions. The majority of these variants were detected below the reported limit of detection of the comparator assays, but above the limit of detection of NS Select. The rate of CHIP mutations was not significantly different in the variants detected by NS Select (19.0% ± 7.6%) vs. comparators (17.2% ± 9.2%). Finally, the proportion of patients with no pathogenic variants detected was shown to be lower by 45% when using NS Select vs. the comparator assays (20/182 vs. 36/182). Conclusions: NS Select reported clinically useful information about the tumor for most patients. For patients in which ctDNA signal was high, NS Select had very high concordance with comparator tests. Furthermore, additional low-VAF pathogenic mutations identified by NS Select translated to a higher diagnostic yield compared to other CGP assays, and CHIP was ruled out as an explanation for these additional detections. This suggests that some patients with low levels of ctDNA would derive clinical benefit from testing with NS Select due to its high sensitivity.

Abstract 5036: Analytical and clinical validity of Northstar Select, a quantitatively enhanced liquid biopsy assay for comprehensive genomic profiling of solid tumors

Abstract Background: Liquid biopsy assays have emerged as vital tools in precision oncology. Among these assays, Comprehensive Genomic Profiling (CGP) tests in particular have improved treatment selection for late-stage solid tumor cancer patients by allowing for the detection via circulating tumor DNA (ctDNA) of somatic mutations in the tumor, many of which have corresponding targeted therapies. However, technical restrictions in the performance of such tests have limited their utility when ctDNA signal is low. BillionToOne’s Northstar Select assay is a new CGP test that achieves increased performance by addressing these limitations. Innovation: By using Quantitative Counting Template (QCT) technology, Northstar Select is able to track the yield of multiple processing steps, contributing to the higher sensitivity of the assay by maximizing the number of decoded molecules after sequencing. In addition, it employs chemistry and panel design optimizations, as well as novel algorithms for SNV, indel, CNV, fusion, and MSI calling to increase sensitivity and specificity. Analytical Performance: The performance of Northstar Select surpasses that of other commercially available tests for multiple mutation types, detecting more pathogenic and actionable mutations at low tumor DNA fraction while maintaining similarly high specificity. In analytical validation (N=103), limit of detection was determined to be 0.13-0.16% variant allele fraction (VAF) for SNVs and indels, 2.11 copies for copy number amplifications, 1.8 copies for copy number losses, 0.35% VAF for fusions, and 0.07% tumor fraction for MSI. Clinical Performance: Northstar Select was determined to have high clinical utility as well, with a study of clinical samples (N=481) from a variety of tumor types yielding <2% of reports with 0 mutations detected. A head-to-head comparison with other commercial liquid biopsy assays on a subset of the clinical samples (N=192) showed that the assay detected 115 additional pathogenic mutations in total, leading to a higher proportion of patients with at least one pathogenic mutation detected (90%) compared to comparators (79%). Conclusion: These results demonstrate that the Northstar Select assay is an innovative and high-performing diagnostic for treatment selection in oncology, leveraging quantitative approaches in process design, chemistry, and bioinformatics to offer superior analytical and clinical performance compared to the current landscape of liquid biopsy CGP tests. Citation Format: Xavier Bower, Jan Wignall, Joyce Zhu, Naomi Searle, Matthew Varga, Emilio Rosas-Linhard, Jason Luong, Esther Lin, Marie Simon, John ten Bosch, Wen Zhou. Analytical and clinical validity of Northstar Select, a quantitatively enhanced liquid biopsy assay for comprehensive genomic profiling of solid tumors [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 5036.

Validation of the Labcorp Plasma Focus Test to Facilitate Precision Oncology Through Cell-Free DNA Genomic Profiling of Solid Tumors

Genomic profiling is critical for precision oncology to guide treatment decisions. Liquid biopsy testing is a complementary approach to tissue testing, particularly when tissue is not readily available. The Labcorp Plasma Focus test is a circulating cell-free DNA genomic profiling test that identifies actionable variants in solid cancers, including non-small-cell lung, colorectal, melanoma, breast, esophageal, gastroesophageal junction, and gastric cancers. This study highlights the analytical validation of the test, including accuracy compared with orthogonal methods, as well as sensitivity, specificity, precision, reproducibility, and repeatability. Concordance with orthogonal methods showed percent positive agreement of 98.7%, 89.3%, and 96.2% for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number amplifications (CNAs), respectively, and 100.0% for translocations and microsatellite instability (MSI). Analytical sensitivity revealed a median limit of detection of 0.7% and 0.6% for SNVs and indels, 1.4-fold for CNAs, 0.5% variant allele frequency for translocations, and 0.6% for MSI. Specificity was >99% for SNVs/indels and 100% for CNAs, translocations, and MSI. Average positive agreement from precision, reproducibility, and repeatability experiments was 97.5% and 88.9% for SNVs/indels and CNAs, and 100% for translocations and MSI. Taken together, these data show that the Labcorp Plasma Focus test is a highly accurate, sensitive, and specific approach for cell-free DNA genomic profiling to supplement tissue testing and inform treatment decisions.

O09: Potential germline variants are frequently identified when performing comprehensive genomic profiling of solid tumors*

O09: Potential germline variants are frequently identified when performing comprehensive genomic profiling of solid tumors*

A scalable high-throughput targeted next-generation sequencing assay for comprehensive genomic profiling of solid tumors.

Timely and accurate identification of molecular alterations in solid tumors is essential for proper management of patients with advanced cancers. This has created a need for rapid, scalable comprehensive genomic profiling (CGP) systems that detect an increasing number of therapeutically-relevant variant types and molecular signatures. In this study, we assessed the analytical performance of the TruSight Oncology 500 High-Throughput assay for detection of somatic alterations from formalin-fixed paraffin-embedded tissue specimens. In parallel, we developed supporting software and automated sample preparation systems designed to process up to 70 clinical samples in a single NovaSeq 6000TM sequencing run with a turnaround time of <7 days from specimen receipt to report. The results demonstrate that the scalable assay accurately and reproducibly detects small variants, copy number alterations, microsatellite instability (MSI) and tumor mutational burden (TMB) from 40ng DNA, and multiple gene fusions, including known and unknown partners and splice variants from 20ng RNA. 717 tumor samples and reference materials with previously known alterations in 96 cancer-related genes were sequenced to evaluate assay performance. All variant classes were reliably detected at consistent and reportable variant allele percentages with >99% overall accuracy and precision. Our results demonstrate that the high-throughput CGP assay is a reliable method for accurate detection of molecular alterations in support of precision therapeutics in oncology. The supporting systems and scalable workflow allow for efficient interpretation and prompt reporting of hundreds of patient cancer genomes per week with excellent analytical performance.

Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors

BackgroundThe translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment. Case presentationThirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLCNOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0–4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases – including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis. ConclusionsCGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.

Functional characterization of SMARCA4 variants identified by targeted exome-sequencing of 131,668 cancer patients

Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy.

Abstract 179: Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus)

Abstract Comprehensive genomic profiling (CGP) of tumor samples by next-generation sequencing is used to support clinical and translational research into the genetic variants that serve as biomarkers for diagnosis, prognosis and potential therapeutic response. However, as these assays grow in size to meet the expanding demands of users, it is challenging to maintain performance in the face of limited sample input necessitated by small sample volumes and to provide a simple and fast sample to report workflow with limited hands-on time. We, therefore, developed OCA Plus to meet user needs for a large CGP assay with excellent performance. Gene content was prioritized based on potential clinical relevance and variant prevalence in solid tumors. Over 500 genes were selected including genes in the indication statements of approved drug labels, clinical guidelines, and in the enrollment criteria of clinical trials. In addition, driver genes were selected in key pathways including DNA repair and immune checkpoint response. Amplicon design strategies were optimized accordingly for key hotspots, full coding sequences or copy number variation (CNV). The assay used Ion AmpliSeq™ technology with manual library preparation or automated templating on the Ion Chef™ System and sequencing on the Ion GeneStudio™ S5 platform. Twenty ng of purified DNA was routinely used as input. An automated tumor-only workflow for variant calling and sample quality reporting was provided within Ion Reporter™ Software. Streamlined access to reporting of variant relevance was enabled by Oncomine™ Reporter. In development studies of cancer cell line and formaldehyde-fixed, paraffin-embedded (FFPE) tumor samples, the assay displayed excellent uniformity (98% and 94%, respectively). Detection of single nucleotide variants and indels in cell lines and FFPE samples showed &amp;gt;95% sensitivity and PPV. Detection of CNV gain and loss in cell lines and FFPE samples showed &amp;gt;95% sensitivity and PPV. Assessment of tumor mutational burden (TMB) using publicly available whole-exome cancer sequencing data as well as test cell lines and FFPE samples showed high concordance with whole exome sequencing (R2 &amp;gt; 0.90). MSI sensitivity and specificity was &amp;gt;95% as tested using a diverse set of tumor samples. Targeted fusions were reported with 100% sensitivity and specificity when tested with commercially available controls. Total time from purified DNA to end of sequencing was &amp;lt; 2 days with &amp;lt; 3 hours of hands-on time and the time from post-sequencing to report generation was &amp;lt; 2 hours. Oncomine Comprehensive Assay (OCA Plus) was developed to support CGP and routine clinical research in oncology. The assay design and informatics workflow were optimized to support low input and rapid sample-to-report turn-around time, which will accelerate clinical and translational research. Citation Format: Vinay Mittal, Jennifer Kilzer, Dinesh Cyanam, Janice Au-Young, Santhoshi Bandla, Gary Bee, Sameh El-Difrawy, Aren Ewing, Rajesh Gottimukkala, Mohit Gupta, Nickolay Khazanov, Anelia Kraltcheva, Amir Marcovitz, Scott Myrand, Rose Putler, Yu-Ting Tseng, Warren Tom, Cristina Van Loy, James Veitch, Paul Williams, Elaine Wong-Ho, Huimin Xie, Chenchen Yang, Zheng Zang, Seth Sadis. Comprehensive genomic profiling of solid tumors for key targeted and immuno-oncology biomarkers using Ion Torrent NGS technology on the Oncomine Comprehensive Assay Plus (OCA Plus) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 179.

Abstract 3652: Analytical validation of a novel circulating tumor DNA detection platform for targeted and immunotherapy selection

Abstract Introduction Comprehensive genomic profiling of solid tumors using circulating tumor DNA (ctDNA) has enabled the detection of all NCCN guideline-recommended somatic genomic classes of alterations from a single, non-invasive blood draw. However, current ctDNA tests still face two major challenges: the inability to reliably identify somatic variants at low mutant allele fraction (MAF), and inconsistency in how the tests have been validated. This study shows how the Single Molecule Sequencing (SMSEQ) platform addresses these challenges. The platform integrates innovative ctDNA extraction methodology, highly optimized library preparation and an error-based variant-calling algorithm to drastically improve sensitivity and specificity. The platform analyzes 5 classes of somatic variants: single nucleotide variants (SNVs), insertions and deletions (Indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Methods We analyzed a 73 gene panel covering NCCN recommended actionable variants for solid tumors in 60 reference ctDNA samples with known variants to establish the limit of detection, sensitivity, specificity, accuracy and reproducibility of the SMSEQ platform. For clinical validation, we tested 36 patients with metastatic colorectal cancer (mCRC) and 34 healthy controls from the Chang Gung Memorial Hospital, and 227 patients diagnosed with solid tumor from Taiwan. Circulating DNA was extracted from plasma followed by library preparation using a highly optimized NGS workflow. Somatic variants in ctDNA are identified using locus-specific modeling to separate tumor variants from normal errors. Results Validation according to recently published ACMG/AMP guidelines shows that the SMSEQ platform allows calling of variants with &amp;gt;99.999% analytical specificity for SNVs, Indels and fusions; and &amp;gt;99% analytical specificity for CNVs and MSI. The platform successfully detected variants at low MAF: 0.1% for SNVs and Indels, &amp;lt;1% for fusions, 5 copies for CNVs, and 1% for MSI. Somatic variants were identified in 35 of 36 mCRC patient samples (97.2%). No false positives were observed within the targeted region for all 34 healthy controls tested. In paired samples, the SMSEQ platform showed 89.7% concordance with tissue biopsy. Observed gene mutation profiles from ctDNA were consistent with published tissue biopsy data: the most frequently mutated genes were TP53, APC, and KRAS; KRAS and BRAF variants were mutually exclusive. In addition to mCRC patients, we tested 227 patients diagnosed with various solid tumors from Taiwan. Actionable variants were detected in 170/227 (75%) patients. Conclusion The CellMax 73-gene liquid biopsy test detects 5 NCCN-guideline recommended variant classes: MSI for immunotherapy and SNVS, Indels, CNVs, and fusions for targeted therapy selection at low variant allele fraction/copy number at high sensitivity and specificity. Citation Format: Pratyush Gupta, Julian Lucas, Alex Atkins, Wen-Sy Tsai, Twinkal Marfatia, Shih-En Chang, Oscar Segurado, Rui Mei. Analytical validation of a novel circulating tumor DNA detection platform for targeted and immunotherapy selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3652.

Confounding effects of clonal hematopoiesis in clinical genomic profiling of solid tumors.

12004Background: Clonal hematopoiesis (CH) is the somatic acquisition of genomic alterations in hematopoietic stem/progenitor cells, leading to clonal expansion. While a few reports showed CH-deriv...

Comprehensive genomic profiling of solid tumors from 677 adolescents and young adults for revealing a distinct spectrum of targetable genomic alterations.

11008 Background: Adolescent and young adult (AYA, age 15-39) cancers (CA) pose unique clincial challenges, yet genomic alterations (GA) in AYA CA have not been widely profiled. To identify potenti...