In breast cancer, the genetic profiling of circulating cell-free DNA (cfDNA) from blood plasma was shown to have good potential for clinical use. In contrast, only a few studies were performed investigating urinary cfDNA. In this pilot study, we analyzed plasma-derived and matching urinary cfDNA samples obtained from 15 presurgical triple-negative breast cancer patients. We used a targeted next-generation sequencing approach to identify and compare genetic alterations in both body fluids. The cfDNA concentration was higher in urine compared to plasma, but there was no significant correlation between matched samples. Bioinformatical analysis revealed a total of 3339 somatic breast-cancer-related variants (VAF ≥ 3%), whereof 1222 vs. 2117 variants were found in plasma-derived vs. urinary cfDNA, respectively. Further, 431 shared variants were found in both body fluids. Throughout the cohort, the recovery rate of plasma-derived mutations in matching urinary cfDNA was 47% and even 63% for pathogenic variants only. The most frequently occurring pathogenic and likely pathogenic mutated genes were NF1, CHEK2, KMT2C and PTEN in both body fluids. Notably, a pathogenic CHEK2 (T519M) variant was found in all 30 samples. Taken together, our results indicated that body fluids appear to be valuable sources bearing complementary information regarding the genetic tumor profile.

Background: Disseminated tumor cells (DTCs) in bone marrow aspirates of patients with primary breast cancer may serve as independent prognostic markers associated with impaired survival. Due to limited therapy options and high risk of recurrence particularly, women diagnosed with the aggressive triple negative breast cancer (TNBC) require personalized treatment choices. Genetic profiling of circulating cell-free tumor DNA (ctDNA) might help to find individual treatment options and to monitor disease course. Methods: Here we report the case of a 66-year-old patient with TNBC. She received neoadjuvant chemotherapy (NACT) that had to be interrupted due to intolerance. Surgical resection of the residual tumor resulted in pathologic complete response (pCR), though. Results: Bone marrow aspiration during surgery revealed an unusual high number of DTCs and thus elevated risk for recurrence. Analysis of pre-surgical blood and urine samples revealed the presence of plasma-derived and urinary ctDNA after NACT and indicated poor prognosis. Subsequent targeted sequencing showed that pathogenic variants occurred in urinary and plasma-derived ctDNA emphasizing the potential of liquid biopsy usage for early detection of relapse. Despite the detection of residual molecular disease after NACT, the presented patient reached pCR and could benefit from standard treatment until present. Conclusions: In this case, liquid biopsy based biomarkers did not necessarily correlate to clinical outcome. Further, ctDNA analysis did not reveal approved therapeutic options to target the identified pathogenic variants. Adjuvant bisphosphonate treatment was applied based on the positive DTC status and may improve the patients' prognosis. Further investigations are required to identify TNBC patients at risk for recurrence.

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. Key words: cfDNA, breast cancer, targeted sequencing, somatic variants, mutation, urine, plasma Citation Format: Henrike Herzog, Bahriye Aktas, Ivonne Nel. Targeted sequencing of plasma-derived vs. urinary cfDNA from patients with triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-16.