Abstract
tRNAscan-SE has been widely used for transfer RNA (tRNA) gene prediction for over twenty years, developed just as the first genomes were decoded. With the massive increase in quantity and ...phylogenetic diversity of genomes, the accurate detection and functional prediction of tRNAs has become more challenging. Utilizing a vastly larger training set, we created nearly one hundred specialized isotype- and clade-specific models, greatly improving tRNAscan-SE’s ability to identify and classify both typical and atypical tRNAs. We employ a new comparative multi-model strategy where predicted tRNAs are scored against a full set of isotype-specific covariance models, allowing functional prediction based on both the anticodon and the highest-scoring isotype model. Comparative model scoring has also enhanced the program's ability to detect tRNA-derived SINEs and other likely pseudogenes. For the first time, tRNAscan-SE also includes fast and highly accurate detection of mitochondrial tRNAs using newly developed models. Overall, tRNA detection sensitivity and specificity is improved for all isotypes, particularly those utilizing specialized models for selenocysteine and the three subtypes of tRNA genes encoding a CAU anticodon. These enhancements will provide researchers with more accurate and detailed tRNA annotation for a wider variety of tRNAs, and may direct attention to tRNAs with novel traits.
Abstract
Background: Molecular response (MR) estimated as a change in circulating tumor (ctDNA) load between an early on treatment sample (usually 2-9 weeks post treatment start) and pre-treatment ...baseline has been shown to predict patient response and outcomes across solid tumors and therapy types in many retrospective studies. There is no consensus, however, regarding the best method for assessing molecular response. Therefore, we aimed to assess several molecular response calculations and determine the optimal method for predicting outcomes in individual advanced cancer patients.
Method: Aggregate results of > 4,000 patient sample pairs (3-10 weeks apart), > 1000 patient sample technical replicates, > 100 contrived sample dilutions, and in silico simulations were analyzed with Guardant360TM or GuardantOMNITM. Baseline and on-treatment paired patient samples were collected from advanced cancer patients with over 12 tumor types, including lung, colon, and breast. MR calculations included variant allele fractions (VAFs) of somatic SNVs, indels and fusions. Methods including Ratio treatment/baseline (R) of Maximum VAF (maxVAF), Ratio of Mean VAF (mVAF), and Mean of VAF Ratios were compared, with consideration of VAF precision. Analytical accuracy, reproducibility and limit of detection (LoD) were assessed.
Results: Comparison of methods for calculating net change in ctDNA load on > 1500 sample pairs showed high correlation (ρ ranged from 0.93 to 0.98) and categorical agreement split by the median (93%). Therefore selecting an optimal method based on outcome prediction would require prohibitively large patient cohorts. Analytical evaluation and in silico simulations can predict the behavior of each method. Simulations of changes in tumor fraction of real pre-treatment samples found that RmVAF or RmaxVAF are more accurate than mVAFR, which can be skewed by low VAF ratios. Almost 25% of sample pairs have a tumor driver or resistance mutation that is not the maxVAF, suggesting tumor dynamics are better captured by mVAF than maxVAF. Newly-detected on-treatment variants can be an important signal of rising ctDNA levels, impacting MR in approximately 2% of sample pairs. Importantly, MR accuracy for all methods decreases as maxVAF approaches or falls below the variant LoD, due to both stochastic detection and higher CV of variants at low VAF. Thus the assay variant LoD is a key determinant of the fraction of patients who can receive MR evaluation. Technical replicates identified the variant criteria at which a 50% change in tumor fraction differs significantly from technical variation, and could define analytical reporting limits.
Conclusions: Comparison of MR methods in a large set of patient samples and simulations supports RmVAF with inclusion of newly-detected mutations. Clinical validation of these methods will support patient-specific MR prediction of outcomes.
Citation Format: Allysia J. Mak, Katie J. Quinn, Carin Espenschied, Kyle Chang, Han-Yu Chuang, Elena Helman, Darya Chudova, Justin Odegaard, Becky Nagy, AmirAli Talasaz. Comparison of molecular response calculations for prediction of patient outcome abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 401.
Abstract
Introduction: Detection of ctDNA in plasma samples permits temporal assessment of tumor mutation status during treatment. Poziotinib is an oral pan-HER TKI that has been demonstrated to be ...efficacious in NSCLC patients harboring HER2 exon 20 insertion mutations. We assessed serial plasma samples for changes in tumor genotype in both treatment naive and second line patients to evaluate correlation with clinical response.
Methods: For NSCLC patients, HER2 exon 20 insertion mutations identified by tumor tissue based NGS were required for entry into the ZENITH20 study. Plasma samples were collected prior to treatment and at C3D1 (8 weeks post treatment 16mg poziotinib QD). The Guardant360® 74-gene liquid biopsy assay, which assessed changes in ctDNA and, subsequently, mean variant allele fraction (mVAF), was utilized for analysis of samples. The Guardant360 Response™ Molecular Response (MR) algorithm was calculated as a ratio of mVAF of oncogenic alterations at baseline compared to poziotinib treatment at C3D1.
Results: 23 patients with tumor tissue confirmed NSCLC harboring HER2 exon 20 insertion mutations were studied. 22 of 23 (96%) had baseline plasma samples with detectable ctDNA. 21 of 22 samples had detectable HER2 exon 20 insertion mutations, resulting in a concordance of 95% versus tissue based NGS. The most prevalent HER2 exon 20 insertion alteration was the A775_G776ins YVMA variant, found in 50% of the baseline blood and tumor samples using both methods (100% concordance). 16 of 17 patients had both baseline and C3D1 samples, permitting assessment of temporal response. 15 of the 16 (94%) patients demonstrated a decrease in mVAF at C3D1 compared to the mVAF at baseline. 12 of 15 patients demonstrated an >50% reduction in mVAF at C3D1, with 7 of the 12 patients showing >95% reduction in mVAF at C3D1 with clinical outcomes of 5 PRs, 1 non-CR/non-PD and 1 SD. Interestingly, 3 of these patients showed complete clearance of ctDNA target HER2 exon 20 insertions at the C3D1 timepoint.
Conclusions: Baseline plasma ctDNA genotyping correlated with tumor tissue based NGS in an NSCLC patient population with HER2 mutations. Poziotinib treatment resulted in mVAF reduction, which correlated with clinical response per RECIST1.1. Assessment of longitudinal changes in ctDNA during drug therapy may potentially be used to predict patient response and possibly tumor resistance. Further evaluation in larger cohorts and longer duration of treatment is required to help elucidate the impact of these findings.
Citation Format: Arunthi Thiagalingam, Sribalaji Lakshmikanthan, Allysia J. Mak, Scott A. Shell, Sharon Leu, Rocky Washington, Lyndah Dreiling, Gajanan Bhat, Francois Lebel, John A. Barrett. Predictive ability of circulating tumor DNA by Guardant360 in poziotinib-treated patients with NSCLC harboring HER2 exon 20 insertion mutations abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3400.
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