Targeted sequencing of plasma-derived vs. urinary cfDNA from patients with triple negative breast cancer
Background: In breast cancer, genetic profiling of circulating cell-free DNA (cfDNA) from blood plasma was shown to have good potential for clinical use and might help to find individual treatment options and to monitor metastatic relapse as well as intratumoral heterogeneity. Due to limited therapy options and high risk of acquired treatment resistance based on genetic alterations of the tumor, particularly, women diagnosed with the aggressive triple negative breast cancer (TNBC) subtype might benefit from serial mutation profiling during disease courses. Here, cfDNA derived from patients' urine might offer promising opportunities for non-invasive monitoring at frequent intervals. In this pilot study, we investigated plasma-derived and matching urinary cfDNA samples obtained from 15 presurgical TNBC patients using a targeted sequencing approach to identify common breast cancer-related genetic alterations in both bodyfluids.  Methods: Blood plasma and urine samples were processed within 4 hrs after collection. We successfully adapted the manufacturer's instructions to yield increased concentrations of extracted plasma-derived and urinary cfDNA (QIAamp MinElute ccfDNA Kit, QIAGEN). After quality control of cfDNA fragments, matched samples were applied to library preparation and target enrichment with a commercially available diagnostic breast cancer specific 93-gene panel service (QIAGEN). Libraries were analyzed by paired-end sequencing on a NextSeq550 (Illumina Inc.) instrument followed by bioinformatical analysis with QIAGEN Biomedical Genomics Workbench and subsequent interpretation of variants using QCI Interpret Translational Software (QIAGEN).  Results: In the cohort, 3339 somatic breast-cancer related genetic alterations (VAF = 3%) were detected, whereof 1222 vs. 2117 variants were found in plasma-derived vs. urinary cfDNA, respectively. Further, 431 variants were found in cfDNA derived from both body fluids. Per sample, we found a median number of 66 variants in plasma vs. 110 variants in urinary cfDNA. Interestingly, a median number of 31 shared variants was found per matched plasma-derived and urinary cfDNA sample resulting in a 46% recovery rate of plasma-derived mutations in urinary cfDNA (and vice versa only a 16% recovery rate of urinary cfDNA variants in plasma) throughout the cohort. Interestingly, QCI analysis revealed that 48 of the mutations found in both body fluids were pathogenic, resulting in a median number of 4 pathogenic variants per sample.  The recovery rate of pathogenic plasma-derived variants in urinary cfDNA was 63% and vice versa, 66% of pathogenic urinary variants could be detected in the matched plasma samples. Further, 33 of the shared mutations were likely pathogenic. Among the patients, the most frequently occurring pathogenic and likely pathogenic mutated genes were NF1, CHEK2, KMT2C and PTEN in both body fluids. Noteworthy, a pathogenic CHEK2 (T519M) variant was found in all 30 samples and two pathogenic NF1 variants (T467I + c.2325+3A>G) as well as a pathogenic PTEN variant (C136Y) were also very frequent in the cohort.  Conclusion: Taken together, our results indicated that both plasma-derived and urinary cfDNA from TNBC patients could be analyzed in a sufficient manner using a targeted sequencing approach. Both body fluids appear to be valuable sources bearing complementary information regarding the genetic tumor profile, which might be relevant for disease monitoring and individual treatment decisions. Further analyses of a larger patient cohort using adequate bioinformatics algorithms are required to find associations between somatic variants in both cfDNA sources.