Abstract 569: Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors
Authors: Tin Antony , Aushev Vasily , Kalashnikova Ekaterina , Salari Raheleh , Shchegrova Svetalana , Fehringer Gordon , Malhotra Meenakshi , Ravi Harini , Sethi Himanshu , Brevnov Maxim , Zimmermann Bernhard , Rodriguez Angel , Billings Paul R. , Aleshin Alexey 
Published: July 2021

Abstract
Introduction: Two metrics are commonly used to quantify tumor-specific variants in cell-free DNA (cfDNA): the variant allele frequency (VAF), defined as the ratio of the number of variant alleles to wild-type alleles, and mean tumor molecules (MTM)/mL of plasma. The main difference between the two metrics is that molecular disease levels expressed in MTMs account for the total amount of cfDNA. Changes in cfDNA levels, which occur in response to cytotoxic therapies, surgery, and inflammation, have shown to impact the detection and quantification of tumor variants in plasma, leading to misinterpretation of the test results. Here we sought to explore the relationship between VAF and MTM and evaluate the performance of each metric in monitoring molecular residual disease.
Methods: We have analyzed 6569 plasma samples from 3389 patients including patients with primary diagnosis of colorectal cancer (n = 2413), breast (n = 170), pancreatic (n = 132), esophageal (n = 86), lung (n = 60), liver (n = 54), other cancers (n = 474) across multiple settings. ctDNA was quantified using a personalized and tumor-informed (bespoke mPCR NGS) assay. We examined the correlation between MTM and VAF for all ctDNA positive plasma samples (n = 1970) from 1283 patients using log-log regression. For patients with longitudinal plasma time points, further analyses on dynamic changes in MTM and VAF was performed to assess the sample-level discrepancy between the two metrics. Furthermore, the absolute values and the dynamic changes in MTM and VAF levels were plotted and compared against clinical truth based on imaging results and patient outcomes.
Results: Across all samples median cfDNA concentrations were 7.9 ng/mL (range: 1.3-381.6 ng/mL). Analysis of all ctDNA positive samples revealed a positive correlation between VAF and MTM, with R2 = 0.91 (slope: 0.86). From 3180 longitudinal plasma samples, we found 2.4% (76/3180) of samples, where changes relative to the previous time point were discordant between MTM and VAF. Interestingly, cfDNA concentrations in these samples were significantly altered. In general, cfDNA levels are representative of intrinsic cell turnover. While VAF is directly related to ctDNA, it is inversely related to cfDNA levels. Increased cfDNA levels were seen in the setting of active cytotoxic therapy, perioperatively, and in the setting of advanced disease, suggesting these settings may be prone for VAF to underestimate the true ctDNA burden. Here we provide evidence that failure of VAF to account for the dynamic changes in cfDNA concentration in plasma may result in misinterpretation of the patient's disease burden. This suggests that in certain indications MTM would be more clinically relevant than VAF.
Conclusion: Although both VAF and MTM are highly correlated, our analysis shows when cfDNA levels are significantly altered, MTM/mL provides a more accurate measure of molecular disease burden.
Citation Format: Antony Tin, Vasily Aushev, Ekaterina Kalashnikova, Raheleh Salari, Svetalana Shchegrova, Gordon Fehringer, Meenakshi Malhotra, Harini Ravi, Himanshu Sethi, Maxim Brevnov, Bernhard Zimmermann, Angel Rodriguez, Paul R. Billings, Alexey Aleshin. Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors [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 569.