Cell-free DNA (cfDNA) is described to mirror intratumoral heterogeneity and gives insight about clonal evolution for improved therapeutic decisions. We sequenced cfDNA of a hormone receptor-positive, HER2-negative metastatic breast cancer (MBC) cohort with a high coverage to examine the prevalence and relevance of any detected variant. cfDNA of 44 MBC patients was isolated, followed by library construction using a customized targeted DNA panel with integrated unique molecular indices analyzing AKT1,

This protocol describes in detail all steps of the library preparation from cfDNA with the QIAseq Targeted DNA Panel (QIAGEN GmbH, Hilden, Germany) and the subsequent data analysis by QIAGEN Biomedical Genomics Workbench and Ingenuity Variant Analysis (both QIAGEN GmbH, Hilden, Germany). Clinically relevant results obtained by this protocol will be published soon. Since the input volume of the cfDNA eluate used in the beginning was 20 µl (and not maximal 16.75 µl as stated in the QIAseq Targeted DNA Panel handbook version Mai 2017), we adjusted the reagent volumes. We used a customized QIAseq Targeted DNA Panel analyzing all exons of the 17 genes of interest (namely AKT1, AR, BRCA1, BRCA2, EGFR, ERBB2, ERBB3, ERCC4, ESR1, KRAS, FGFR1MUC16, PIK3CA, PIK3R1, PTEN, PTGFR, TGFB1). In contrast to the QIAseq Targeted DNA Panel handbook, we used 20 cycles of amplification for the universal PCR. For stringent data analysis, we defined criteria to exclude libraries with unsufficient sequencing qualities and we here also describe the bioinformatical filter used within the Ingenuity Variant Analysis software (QIAGEN GmbH, Hilden, Germany) for variant exclusion.

Background: The detection and characterization of circulating tumor cells (CTCs) as one of the analytes in liquid biopsy has been considered as surrogate marker to improve treatment decisions in metastatic breast cancer (MBC). In addition, cell-free tumor DNA (ctDNA) released by tumor cells and harboring tumor-associated variants is further discussed to give additional information for therapeutic options. Thus, CTC and ctDNA analysis from the same blood tube is desired. To test usability of plasma, generated after CTC isolation from whole blood for ctDNA analysis, we analyzed ctDNA from 42 hormone receptor-positive/HER2-negative MBC patients (pts) for the detection of tumor-associated variants (plasma isolated straight from whole blood) and compared the results for similarities and differences of the detected variants in a subgroup of these pts to those, obtained from plasma generated after CTC selection (taken from a separate tube). Methods: 4 ml plasma of all MBC pts and 4 ml plasma obtained after immunomagnetic isolation of CTCs from 2x5ml blood [AdnaTest EMT-2/Stem Cell Select (n=17pts) followed by multimarker qPCR] were used for the analysis of cell-free DNA (cfDNA) applying the QIAamp MinElute ccfDNA Kit. A total of 30ng - 60ng cfDNA was applied for library construction using the QIAseq Targeted DNA Panel for Illumina with integrated unique molecular identifiers. Sequencing was executed on the NextSeq® 500 platform (Illumina, US). Data were analyzed using the QIAseq Targeted Sequencing Data Analysis Portal, the Biomedical Genomics Workbench and the Ingenuity Variant Analysis. All materials used were manufactured by QIAGEN, Germany. Results: In the total cohort of 42 pts, most variants of all analyzed genes were detected in the MUC16 gene (31.2%). ERBB2, EGFR and AR (androgen receptor) also showed high numbers of variants (11.6%, 11.0% and 8.9%, respectively) with a majority detected pathogenic variants (47.7%) in AR. 92% of all detected variants showed an allele frequency of <5% and some of the detected MUC16, ERBB2 and AR mutations significantly correlated with overall survival. Comparing the plasma results from a separate blood draw with the results from plasma samples after CTC selection in a subgroup of 17/42 pts, no significant difference was found for cfDNA concentration but variability within the cohort. Whereas the variant comparison of ctDNA isolated from both plasma sources showed great concordance, additional variants (around 15%) were exclusively found in one of the two matched samples. Interestingly, in the variant population exclusively found in ctDNA isolated after CTC isolation, the relative amount of pathogenic variants was increased compared to the variant fraction only found in ctDNA from plasma of a separate blood tube. Results obtained for frequently overexpressed CTC transcripts in this subgroup included genes involved in the PI3K signaling pathway as well as ERBB2 and ERBB3 in about 30% of the pts. Conclusion: We here present a feasible workflow for CTC and ctDNA evaluation for expression and mutation analysis from the same blood sample. These data emphasize that the use of different liquid biopsy analytes can empower treatment decisions of MBC pts in the future. Citation Format: Kasimir-Bauer S, Bittner A-K, Hoffmann O, Hauch S, Sprenger-Haussels M, Storbeck M, Benyaa K, Hahn P, Mach P, Tewes M, Kimmig R, and Keup C. The analysis of cell-free DNA and circulating tumor cells from one blood tube might empower treatment decisions in metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-10.

Background: Blood analytes, as liquid biopsies, are discussed to be surrogate markers for therapy stratification, as serial sampling enabled by the minimal invasive nature of blood draw facilitates monitoring of clonal evolution. Mutational analysis of cell-free DNA (cfDNA) in plasma of breast cancer (BC) patients (pts) may predict the ideal therapy strategy. In this regard, PIK3CA mutations in BC pts' cfDNA were already shown to correlate with treatment response. However, in Next Generation Sequencing (NGS) workflows, used for cfDNA analysis, artefacts are often induced during library construction. Here we used unique molecular identifiers (UMIs) to verify true positive PIK3CA mutations in cfDNA of BC pts. Patients and methods: cfDNA was isolated from 54 plasma samples of 38 BC patients by affinity-based binding to magnetic beads (QIAamp MinElute ccfDNA Kit, QIAGEN, Germany). 30ng - 60ng cfDNA was used for library construction with the QIAseq Targeted DNA Panel for Illumina (QIAGEN, Germany) with integrated UMIs. Sequencing was executed on the NextSeq ® 500 platform (Illumina, US). Data analysis was performed by QIAseq Targeted Sequencing Data Analysis Portal and the Biomedical Genomics Workbench. As a reference, the PIK3CA mutational status of matched tumor tissue DNA (analyzed by Sanger sequencing) was consulted. Results: Library preparation was successful (yield >15ng) in 52/54 cases. Mean coverage was ~20.000x (mean UMI coverage ~2.500x) and >10.000x in 48/52 cases. The minimal allele frequency found for PIK3CA hotspot mutations (P539S, E545K, H1047R) by UMI analysis was 0.72%. In total, 133 mutations of the PIK3CA gene were identified as true positive mutations by UMI analysis in all 52 samples, which is a reduction of 69% (294/427) of all PIK3CA mutations incorrectly called by conventional data analysis. 59% of all different PIK3CA mutations called by UMI analysis appeared in over 5% of all pts. In the cohort of pts with PIK3CA hotspot mutant tumor tissue, 32% (6/19) showed the mentioned mutations also in matched cfDNA, whereas 16% of pts (3/19) without PIK3CA mutant tumor tissue were identified with true positive PIK3CA hotspot mutations in their plasma. Longitudinal analysis across two years during therapy revealed the increase in allele frequency (0%;11%;39%) of the PIK3CA H1047R mutation in one pt, whereas another pt showed a stable allele frequency of the PIK3CA P539S mutation (52%; 51%; 48%). Results will be expanded by consideration of mutations in BC hotspot genes despite PIK3CA and correlation to clinical parameter. Conclusions: Unique molecular identifiers enable the identification of true positive mutations in cfDNA and can thus, be used in clinical practice to determine molecular drivers of individual cancer progression and to employ personalized therapy. Citation Format: Corinna Keup, Karim Benyaa, Peter Hahn, Siegfried Hauch, Pawel Mach, Mitra Tewes, Hans-Christian Kolberg, Sabine Kasimir-Bauer. Use of unique molecular identifiers to gain insight about the true positive mutations in cfDNA of breast cancer patients for implementation of personalized treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3650.