Abstract 3105: Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma
Authors: Bosse Kristopher R. , Buongervino Samantha , Lane Maria , Hyman Adam , Gemino-borromeo Maria , Saggio Jennifer , Fitzsimmons Alana , Forcier Brady , Murphy Anne , Wick John , Cooke Matthew , Webster Jennifer , Madison Russell , Welsh Alley , Miller Vincent A. , Ali Siraj M. , Maris John M. , Mosse Yael P. 
Published: August 2019

Abstract
Background: Analysis of circulating tumor DNA (ctDNA) provides a noninvasive method to profile tumor-associated genomic aberrations, heterogeneity and evolution.
Methods: Peripheral blood from 5 newly diagnosed and 33 relapsed high-risk neuroblastoma patients was serially profiled for ctDNA (n=1-7 samples/patient; total n=122 samples) with the FoundationACT assay that utilizes hybrid capture-based genomic profiling of 62 genes. Samples were sequenced to a median unique coverage depth of at least 3168x and variants were evaluated and compared with temporally-matched tumor sequencing and imaging evaluations.
Results: Ninety-three percent (114/122) of peripheral blood samples yielded suitable cell-free DNA for sequencing. ctDNA was detected in 68% (78/114) of samples (maximum somatic allele frequency [MSAF] &gt;0; median MSAF=0.53%; range MSAF 0-79%). At least 1 pathogenic genomic alteration (genomic short-variant or amplification) was found in 54% (61/114) of samples (range 1-6 alterations). Fifty-four percent (13/24) of detected ctDNA genomic-short variants were not present in temporally matched tumor samples at diagnosis or relapse (collected within 3 months of each other; n=23 patients), including pathogenic variants in ALK, TP53, TERT, NF1, FLT3, PTPN11, and PIK3CA. There was 100% concordance between the detection of MYCN (6/6) and ALK (3/3) amplification in paired ctDNA/tissue samples. For example, a newly diagnosed patient with stage 4 neuroblastoma had MYCN amplification noted on both tumor and ctDNA sequencing, however had 3 separate ALKmutations (R1275Q, F1245L, and F1174L) uniquely identified in ctDNA. Twenty-eight patients had multiple ctDNA samples sequenced (range 2-7 samples) and 50% (14/28) had alterations emerge or regress across serial samples. For example, pathogenic variants in ALK, BRCA2, NRAS, PTEN, TP53, CDKN2A, PTPN11, ABL1,CDH1, MET, ERRFl1 and ERBB2 appeared in subsequent ctDNA sequencing that were not present in the initial ctDNA or tumor sequencing. Overall, serial ctDNA profiling identified additional pathogenic variants in driver cancer genes beyond that derived from tumor sequencing in 45% (17/38) of cases, including identifying targetable ALK-RAS-MAPK pathway alterations uniquely in ctDNA in 21% (8/38) of cases. For the 18 patients that had 3 or more ctDNA samples, 33% (6/18) developed ctDNA unique ALK-RAS-MAPK pathway mutations during therapy. Finally, in most cases ctDNA profiling was complementary to standard imaging surveillance, however in 3 cases rising ctDNA allele frequencies occurred prior to clinical or imaging signs of disease relapse; additional correlation of ctDNA data and disease evaluations is ongoing.
Conclusions: Sequencing of ctDNA from neuroblastoma patients identified clinically actionable tumor-associated genetic aberrations emerging under the selective pressure of standard and targeted therapies.
Citation Format: Kristopher R. Bosse, Samantha Buongervino, Maria Lane, Adam Hyman, Maria Gemino-borromeo, Jennifer Saggio, Alana Fitzsimmons, Brady Forcier, Anne Murphy, John Wick, Matthew Cooke, Jennifer Webster, Russell Madison, Alley Welsh, Vincent A. Miller, Siraj M. Ali, John M. Maris, Yael P. Mosse. Serial profiling of ctDNA identifies clinically actionable genomic evolution in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3105.