Background: Circulating tumor DNA (ctDNA) level and the change in level at a subsequent time point (e.g. on-treatment change from baseline or postoperative changes through time) are promising tools for predicting patient prognosis and response to therapy. Existing methods use somatic variant allele frequencies to quantify circulating tumor fractions (cTF). Their performance can be limited by the number of detectable somatic alterations and the associated limit of detection (LoD), as well as interference from copy number variation and non-tumor alterations, such as clonal hematopoiesis. Here, we describe the LoD, precision and limit of quantitation (LoQ) of cTF level and change using GuardantINFINITY, a next generation sequencing panel covering over 800 genes with genome-wide methylation detection. Method: The cTF of a single sample is estimated from methylation signals across targeted regions of the GuardantINFINITY methylation panel, calibrated using internal training data. cTF change compares two or more samples from the same patient to identify patient-specific methylated regions, and compare the methylation signals of the paired regions. LoQ of cTF level and change were assessed in experimental titrations of advanced colorectal, breast, and lung cancer patient samples and cell line samples into cancer-free backgrounds at different target levels between 0.1%-0.5% cTF. LoD is defined as the lowest cTF level where >95% replicates were detected to have tumor-derived methylation signals. LoQ of cTF level or change is defined as the lowest cTF where the coefficient of variation (CV) across replicates is less than 30%. Accuracy of methylation based cTF compared to cTFs calculated from maximum VAF of somatic mutations was assessed on 1,400 clinical samples of colorectal and lung cancer patients (N=189, 372, 252 and 463 for stage I to IV). Results: Experimental titrations of cancer samples demonstrated a single-sample LoD of 0.05% cTF (lowest dilution level) and quantitative precision down to a LoQ of below 0.1%, compared to the LoQ of 0.3% estimated by somatic mutations. In paired clinical titration samples, the LoQ of methylation ctDNA level change was also below 0.1%, compared to the LoQ of ctDNA level change estimated by somatic mutations at 0.3-0.5%. In the 1,400 clinical samples, 64% had at least one somatic mutation detected, 90% had ct-DNA detected with methylation and 96% of these ct-DNA detected samples had cTF above the defined methylation LoQ. Among patients with both methylation and genomic signals identified, the methylation method quantified a similar cTF to those that were calculated using maximum somatic driver mutations (Pearson r=0.83). Conclusion: Methylome sequencing using GuardantINFINITY enables accurate and precise quantification of ctDNA level and change with a liquid-only approach, offering longitudinal ctDNA monitoring for more patients than previous methods. Citation Format: Sai Chen, Katie Quinn, Che-Yu Lee, Jun Zhao, Kyle Chang, Tingting Jiang, Shile Zhang, Carin Espenschied, Sara Wienke, Thereasa Rich, Indira Wu, Yvonne Kim, Xianxian Liu, Nageswara Alla, Dustin Ma, Giao Tran, Han-Yu Chuang. A method for quantifying circulating tumor DNA level and molecular response using methylome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3123.
Background: Despite its revolutionary impact, cancer genomics alone provides little information on tumor phenotype or functional state, which are governed by epigenetic mechanisms, notably methylation of regulatory regions. Tumor and host epigenetic methylation signatures reflect not only tumor phenotype, such as histology, prognosis, protein expression, and functional sub-type, but also that of the tumor microenvironment and the patient, including immune status, therapy-related adverse events, comorbidities, and disease location. Epigenetic markers also provide more sensitive and precise measures of tumor burden, opening up applications for longitudinal therapy response and monitoring. Here we report the initial validation of GuardantINFINITY, a liquid biopsy assay combining genomic information from >800 genes with characterization of the blood-quiet regulatory methylome, both at single-molecule sensitivity from a single tube of peripheral blood. Methods: Analytical performance was assessed using 594 cancer patient cfDNA, cell line, and cancer-free donor samples at 5-30ng cfDNA input. Results: Reportable ranges established for SNVs were ≥0.04% variant allele fraction (VAF), ≥0.04% for indels, ≥0.06% for fusions, ≥2.12 copies for amplifications (CNAs), <1.7 copies for copy loss. Observed 95% limits of detection (LoD) were 0.282% for SNVs across all genes (0.2% for oncogenic hotspots), 0.397% for non-homopolymeric indels, 0.05% for fusions, 2.5 copies for CNAs, 16.3% VAF or 1.84 copies for gene deletions, 7.3 copies for viral (HPV, EBV) detection, and 0.06% for MSI-H. For promoter and sample-level methylation, LoDs were 0.06% and 0.05% tumor fraction, respectively. cfDNA cancer samples demonstrated 100% accuracy for SNVs and Indels above 0.5% VAF and 100% for CNAs and fusions across the reportable range. The analytical false positive rate per base was 6.84e-6 for SNVs, 3.42e-6 for indels, and 0 for CNAs and fusions, with positive predictive values of 97.5% for SNVs, 98% for indels, and 100% for CNAs above 2.5 copies and all tested fusions. Conclusions: GuardantINFINITY is a patient-care-ready liquid biopsy capable of integrated genomic and epigenomic analysis of all solid tumors at single-molecule sensitivity. In addition to traditional genotyping compatible with Guardant360 for more content, the technology’s demonstrated LoD showed the potential for ultra-sensitive ctDNA detection for MRD and recurrence surveillance, tumor fraction quantitation for therapy monitoring, oncogenic virus detection, immunogenotyping, epigenotyping, and tumor phenotype characterization, representing a new standard in biomarker discovery. Citation Format: Tingting Jiang, Indira Wu, Yvonne Kim, Nageswara Alla, Giao Tran, Dustin Ma, Forum Shah, Jun Zhao, Sai Chen, Sante Gnerre, Melis Hazar, Hao Wang, Catalin Barbacioru, Karen Ryall, Ankit Jambusaria, Anupam Chakravarthy, Anthony Zunino, Theresa Pham, Farsheed Ghadiri, Evan Diehl, Benjamin Morck, Arancha Sanchez, Rochelle Dayan, XianXian Liu, Jeffrey Werbin, Jill Lai, Brett Kennedy, Ross Eppler, Justin Odegaard, Han-Yu Chuang, Helmy Eltoukhy. Analytical validation of a robust integrated genomic and epigenomic liquid biopsy for biomarker discovery, therapy selection, and response monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6601.
Background: HLA germline genotypes and somatic mutations show great promise as emerging biomarkers for immune checkpoint inhibitors (ICIs) and understanding patient prognosis. Multiple studies have shown that HLA homozygosity or loss-of-function somatic mutations negatively correlates ICI response rate. Here we present additional data on the algorithm Kmerizer, designed to perform HLA germline typing and somatic mutation detection from cfDNA input material, and we show how we use these in neoantigen prioritization for patient outcome prediction. Methods: Kmerizer first leverages the high depth coverage of targeted sequencing to rapidly identify germline alleles by matching k-mers from the input reads to the k-mers of known HLA alleles. Careful realignment of reads ontothe called germlines is followed by proprietary somatic variant calling. MHC class1 germline allele calls are combined with patient mutation data to generate in silico TCR binding affinity predictions using net MHC-4.0. These predictions are compared across cohorts to assess how cancer type, TMB, and ICI response vary with the predicted neoantigens and TCR binding affinity. Results: Of nineteen cell lines, twelve plasma samples and eight gDNA samples with confirmed HLA typing information, Kmerizer delivered 100% sensitivity on both MHC-I and II genes, with 99.5% and 98.7% specificity, respectively, based on GuardantINFINITY cfDNA sequencing data. For homozygous/heterozygous status, accuracy of 99.1% on class I and 97.7% on class II genes is achieved. HLA allele prevalence among our development samples is consistent with reference cohorts of similar geographic origin in MHC class I genes. The HLA somatic caller achieves >99.99% specificity per base as computed on 48 normal samples, while achieves >91% sensitivity for somatic events with expected allele frequency (AF) ~ 0.15% (AF range[0.08%,0.26%] for detected events) as evaluated through simulations. Additionally, we generated a total of 2,767 immunogenic (ic50<500nM) class-I somatic neoantigens predictions across 112 samples from cancer patients with germline HLA typing results. We found average patient neoantigen TCR binding affinity was significantly associated with cancer type (χ2=86.08,p<0.0001). Top predicted neoantigen binding affinity across patient HLA types were strongly inversely correlated with patient bTMB(rhospearman=-0.25, p<0.0001). Conclusions: The integration of Kmerizer into GuardantINFINITY enables accurate HLA germline and somatic detection along with neoantigen prediction, offering an enhanced and comprehensive biomarker profiling for ICI outcome prediction. Citation Format: Sante Gnerre, Jun Zhao, Adrian Bubie, Yvonne Kim, Dustin Ma, Indira Wu, Bojan Losic, Tingting Jiang, Han-Yu Chuang. Using Kmerizer, a germline and somatic genotyper for immune associated complex alleles in GuardantINFINITY, for immunotherapy response prediction using cfDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3128.
Background: BRCA1 promoter methylation (PM) is an early initiating event in cancer, occurring in 3 to 65.2% of all breast tumors, and 30 to 65% of triple negative tumors. BRCA1 PM has been associated with defective homologous recombination repair (HRR), early onset of breast and ovarian cancer, and improved clinical response to adjuvant chemotherapy. Historically, there has been no diagnostic assay that comprehensively evaluates both BRCA1 PM and genomic alterations in cell-free circulating tumor DNA (ctDNA). Here, we describe the novel detection of BRCA1 PM and genomic alterations in a cohort of patients with breast cancer using GuardantINFINITY, a liquid biopsy assay interrogating 800+ genes and genome-wide methylation detection. Method: We assessed for BRCA1 PM in ctDNA from 274 patients with late-stage breast cancer. Genomic sequencing of 800+ genes and PM profiling of 398 genes was performed by GuardantINFINITY. The positive calling threshold for PM was established by comparing cell-free DNA derived from patients with cancer and cancer-free donors. The limit of detection (LoD) was determined through in silico and experimental titrations of ctDNA from clinical samples and cell lines with known gene PM into the plasma of cancer-free donors. Results: Among the 274 patients with advanced breast cancer, 8 (2.9%) had germline pathogenic mutations in BRCA1, BRCA2, or ATM. BRCA1 PM was detected in 11/274 (4.0%) patients at the predefined threshold of >99% specificity. BRCA1 PM detection in this cohort was 8.9% (8/90) when excluding samples with low tumor shedding (<1% epigenomic tumor fraction in cfDNA). Among the 11 patients with BRCA1 PM detected in ctDNA, one had a co-occurring somatic BRCA1 nonsense variant (p.S361*); none of the remaining patients with BRCA1 PM had another HRR-related mutation detected in cfDNA. Among patients without BRCA1 PM detected, pathogenic somatic alterations were detected in BRCA2, ATM, and CHEK2 in 25 (9.4%) patients. In silico simulations using clinical samples with BRCA1 PM indicated an LoD of 0.0408%. BRCA1 PM was not detected in 3210 individual and mixed cancer-free clinical samples, indicating a high specificity for BRCA1 PM calls. Conclusion: GuardantINFINITY, a plasma-based diagnostic assay, detected both BRCA1 PM and genomic alterations in this unspecified advanced breast cancer cohort. The BRCA1 PM detection rates of 4.0-8.9% are consistent with values previously reported in the literature. As BRCA1 PM has important prognostic and therapeutic implications for the management of breast (as well as ovarian) cancers, additional studies are warranted to further describe the PM patterns across breast cancer subtypes and how these patterns both influence and are influenced by disease evolution and therapeutic response. Liquid biopsy thus serves as a suitable method to noninvasively identify and monitor changes in both genomics and epigenomics. Citation Format: Jennifer Yen, Sai Chen, Colby Jenkins, Brooke Overstreet, Yu Fu, Jun Zhao, Tingting Jiang, Leylah Drusbosky, Stephen Pettitt, Michael Dorschner, Lauren Lawrence, Han-Yu Chuang, Andrew Tutt. BRCA1 promoter methylation in sporadic breast cancer patients detected by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6603.
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