Abstract A05: Circulating tumor DNA levels correlate with response to treatment in LMS patients
Authors: Przybyl Joanna , Chabon Jacob J. , Spans Lien , Ganjoo Kristen , Vennam Sujay , Newman Aaron M. , Forgó Erna , Varma Sushama , Zhu Shirley , Debiec-Rychter Maria , Alizadeh Ash , Diehn Maximilian , Rijn Matt van de 
Published: August 2018

Abstract
Circulating tumor DNA (ctDNA) has significant potential for several clinical applications, including assessment of treatment response and monitoring of recurrent/residual disease. We performed a pilot study to explore the feasibility of ctDNA monitoring in patients with leiomyosarcoma (LMS).
We profiled matching plasma and FFPE tumor specimens from 9 LMS patients. We analyzed between 2 to 6 longitudinal plasma samples (median of 5) and between 1 to 7 tumor specimens (median of 2) per patient. ctDNA analysis was performed on plasma samples collected pre-/post-surgery, throughout chemo-/radiotherapy and during follow-up. We used two separate approaches in our study: 1) targeted deep sequencing of ctDNA, tumor DNA and germline DNA to detect single nucleotide variants and indels using Cancer Personalized Profiling by deep Sequencing with integrated digital error suppression (CAPP-Seq; with a median deduplicated depth of sequencing of 2,136x); 2) copy number variant analysis in ctDNA by genome representation profiling (GRP; median coverage across the whole genome 0.23x) and in the matched tumors by SNP arrays. One patient was excluded from the analysis due to inadequate sequencing coverage in tumor specimen.
For CAPP-Seq analysis, we designed a custom 184kb capture panel targeting 89 genes that are recurrently mutated in LMS. Using strict variant calling criteria (requiring that variants be present on each strand of the original DNA “duplex” molecule) our panel identified a median of one nonsynonymous coding/splicing variant per tumor. We detected the same variants in TP53, RB1 and ATRX genes in ctDNA of 6/8 patients (with a baseline sensitivity of 87.5% and overall specificity of 98.96% calculated using plasma from 24 healthy donors). These six patients presented with advanced disease at the time of the first blood collection and were progressing throughout multiple lines of therapy. Two patients who did not have any variants detectable by CAPP-Seq in plasma had localized disease at the time of the first blood collection and/or responded well to the therapy. We found that changes in ctDNA levels appear to correspond with the extent of disease and response to treatment. Specifically, ctDNA levels decreased in a subset of patients after surgery or at the time of temporary response to chemo- and/or radiotherapy. Congruently, increases in ctDNA levels correlated with progression in most of the patients. There was a high correlation between ctDNA levels detected by CAPP-Seq (quantified as mutant molecules/mL plasma) and GRP (quantified as percent of genome showing copy number aberrations) across all plasma samples (Pearson's r= 0.88, p &lt; 0.0001), but in a few samples ctDNA was detected by only one of the two assays.
Our results suggest that serial analysis of ctDNA is a promising approach for evaluation of treatment response in LMS patients. Validation of these findings in a prospective study on a larger group of patients will be required to determine the use of this approach in a clinical setting.
References:
CAPP-Seq: PMIDs 24705333, 27018799
GRP: PMIDs 25585704, 26687610
Citation Format: Joanna Przybyl, Jacob J. Chabon, Lien Spans, Kristen Ganjoo, Sujay Vennam, Aaron M. Newman, Erna Forgó, Sushama Varma, Shirley Zhu, Maria Debiec-Rychter, Ash Alizadeh, Maximilian Diehn, Matt van de Rijn. Circulating tumor DNA levels correlate with response to treatment in LMS patients [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A05.