Unique Molecular Index Research Articles

Abstract 816: Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies

Abstract A clinical next generation sequencing (NGS) assay comprises various factors taken into consideration including selection of appropriate markers thereby panel size, nucleic acid input, assessment and interpretation, and workflow. The downstream benefits of the development of such panels therefore involve improvement in prognosis, patients' outcome, and benefit from targeted therapy. Myeloid malignancies such as myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN, acute myeloid leukemia (AML) are associated with somatic mutations in around 25 to 50 genes. Herein, we describe clinical validation of a DNA based targeted NGS assay utilizing QIAact Myeloid DNA UMI panel in combination with QIAGEN GeneReader NGS system. The assay employs unique molecular index technology (UMI) into a gene specific, primer based target enrichment technology enabling sequencing of specific regions of interest providing an integrated solution by simultaneously assessing many candidate genes for actionable mutations. The QIAact Myeloid DNA UMI panel is a 25 gene panel for markers of known clinical significance, including single nucleotide variants (SNV), and large insertion/deletion mutations. At the lab, 40 individual AML specimens with known mutation profiles were tested. The limit of detection (LOD) was assessed by sequencing Seraseq myeloid mutation DNA mix as positive control. For accuracy, inter-run and intra-run controls were also implemented. All samples were previously characterized by using another 54 gene NGS panel. The samples were prepared using QIAact Myeloid DNA UMI panel kits, sequenced with QIAGEN GeneReader NGS system, and mutations identified using the QIAGEN Clinical Insight (QCI) Analyze software suite, adjusted specifically for variant calling. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection (LOD) studies also determined that input of 40ng DNA was optimal for high analytical sensitivity. High positive and negative percentage agreement with prior results was observed across all variant categories. The assay was also able to identify 52bp deletion CALR type 1 variant, and CEBPα amplicons mutations which require separate bi-directional Sanger sequencing for identification. Also, the assay was able to identify FLT3 ITDs up to 101bp insertion which are detected by performing PCR separately. Hence, the study presents the data establishing the QIAact Myeloid DNA UMI panel suitable for implementation as a routine clinical NGS test for myeloid malignancies. The optimized chemistry allows high-throughput analytical sensitivity for detection of highly relevant mutations including large Insertion/Deletion (InDel). Citation Format: Meenakshi Ahluwalia, Ashis Mondal, Nikhil Sahajpal, Allan Njau, Kimya Jones, Pankaj Kumar Ahluwalia, Yasmeen Jilani, Ravindra Kolhe. Utility and validation of a comprehensive cost-effective targeted DNA panel including FLT3-ITDs, CALR and CEBPA on a next-generation sequencing (NGS) platform for hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 816.

Abstract PR08: Validation and clinical implementation of MSK-ACCESS, an ultra-deep sequencing assay for noninvasive somatic mutation profiling

Abstract Introduction: Circulating cell-free DNA (cfDNA) is a source of tumor-derived DNA to interrogate somatic alterations when tissue is not available or of insufficient quantity for analysis. At MSKCC, we have developed and validated MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), a targeted next-generation sequencing assay that can detect ultra-low frequency somatic variants in select exons and introns of 129 genes. MSK-ACCESS can identify mutations, copy number alterations, gene fusions, and MSI status in plasma and was recently approved by the NYS-DOH for clinical testing. Here, we present the results of the validation study and our clinical experience with MSK-ACCESS since June 2019. Methods: Target regions from 129 genes were selected to maximize coverage of actionable, oncogenic, and hotspot mutations based on the first 25,000 tumors sequenced using MSK-IMPACT, our institutional clinical sequencing assay. Plasma cfDNA and buffy-coat DNA were extracted from whole blood collected in cell-stabilizing tubes (STRECK BCT cell-free DNA tube). Unique molecular indexes were introduced during DNA library construction, allowing for error suppression from consensus reads collapsed by Marianas, an in-house-developed algorithm. These consensus reads enable variant calling at low allelic frequency (AF) based on a 10−6 background error rate. Results: Analytical validation of MSK-ACCESS demonstrated 93% accuracy (n=100 variants), 99% precision (n=153 variants), and 100% sensitivity based on an assay limit of detection of 0.5% AF (n=19 variants). Variants were detected down to 0.1% AF. To date, 240 clinical cfDNA and matched normal DNA pairs have been sequenced, analyzed for somatic alterations, and clinically reported to guide patient management. Most clinical cases were from lung (55%) or prostate (13%) cancers and submitted for diagnostic purposes (71%). Median raw coverage was 18,367X, and median consensus coverage was 1411X. Mutations were detected in 180 (75%) samples with a median variant AF of 1.8% (0.02% - 95%). Comparison of concurrent commercial plasma testing results to MSK-ACCESS revealed multiple variants that were of clonal hematopoiesis or germline origin incorrectly reported as somatic variants. In the lung cohort, 48 patients had tissue testing with MSK-IMPACT; among 32 patients with a driver alteration detected by MSK-ACCESS, 91% had the identical driver alteration reported by MSK-IMPACT. Additionally, MSK-ACCESS identified a MET exon 14 alteration in one lung cancer patient that led to protocol enrollment and partial response. Conclusions: Liquid biopsy testing using MSK-ACCESS reliably detected clinically actionable mutations, reducing the need for multiple biopsies. These results also illustrate the importance of analyzing a matched normal sample when interpreting cfDNA results and highlight the potential of using cfDNA analysis to guide treatment selection, assess for treatment response, and identify mechanisms of treatment resistance. This abstract is also being presented as Poster A20. Citation Format: A. Rose Brannon, Gowtham Jayakumaran, Monica Diosdado, Yu Hu, Anna Razumova, Fanli Meng, Emily Lebow, Juber Patel, Ian Johnson, Preethi Srinivasan, Maysun Hasan, Jenna-marie Dix, Aijazuddin Syed, Brian Houck-Loomis, Bob T. Li, Charles Rudin, David Solit, Marc Ladanyi, Maria Arcila, Dana Tsui, Ahmet Zehir, Michael Berger, Ryma Benayed. Validation and clinical implementation of MSK-ACCESS, an ultra-deep sequencing assay for noninvasive somatic mutation profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR08.

Abstract A29: Next-generation sequencing of circulating tumor DNA to monitor treatment response to CDK4/6 inhibitors in breast cancer

Abstract Background: CDK4/6 inhibitors are effective treatment modalities against hormone receptor-positive metastatic breast cancer (MBC). However, monitoring of clinical responses relies solely on conventional tumor markers that are insufficiently sensitive and specific as well as imaging methods that could not be performed regularly. In this study, we aimed to evaluate the potential utility of circulating tumor DNA (ctDNA) as a complementary approach to monitor clinical response and to evaluate resistance mechanisms associated with CDK4/6 treatment in MBC. Method: The monitoring duration of this study is up to 24 months or until patient develops disease progression. In this interim report, a total of 100 samples from 21 MBC patients who received CDK4/6 inhibitors were collected before and after treatment. Cell-free DNA/RNA was extracted from plasma and subjected to ultradeep targeted next-generation sequencing (NGS) using Oncomine Pan-Cancer cell-free assay that incorporates the use of unique molecular index. This panel covers more than 1,000 hotspot mutations, 12 CNVs, and 12 fusions in cancer-related genes. Paired NGS was performed with genomic DNA from buffy coat to exclude clonal-hematopoiesis derived mutations. The changes in mutation allele frequency of ctDNA will be compared with tumor markers CA15-3 and CEA as well as CT-imaging and PET-scan results. Results: According to RECIST criteria, one patient showed complete response 9 months after start of treatment while 11 patients showed partial response or stable disease within 3-12 months from start of treatment. A total of 9 out of 21 MBC patients developed progression of disease within 9 months of treatment. The total and molecular depth of targeted NGS are ~50,000X and ~4000X, respectively. A total of 20 nonsynonymous mutations and 5 copy number variations (CNV) were detected in 16 of the 21 patients (76%) before treatment for response monitoring. The majority of the mutations are in TP53, PIK3CA, and ESR1 genes. The ctDNA profiles were concordant with clinical responses in the majority of the patients. Mutation allele frequencies of ctDNA showed better dynamic changes compared with tumor biomarkers CEA and CA15-3. NGS of ctDNA was able to detect disease progression 3 months in advance or concurrent with imaging data in this study. Diverse ctDNA profiles were observed, indicating the usefulness of ctDNA in detecting intra- and intertumoral heterogeneity. We observed acquired mutations in AKT1 and PIK3CA in one patient, suggesting these mutations were potential CDK4/6 resistance mutations. Conclusion: Our results suggest that ctDNA may be applied as a robust biomarker to monitor clinical response to the CDK4/6 treatment. Citation Format: Yoon Ming Chin, Tomoko Shibayama, Masumi Otaki, Hiu Ting Chan, Makiko Ono, Yoshinori Ito, Shunji Takahashi, Shinji Ohno, Takayuki Ueno, Yusuke Nakamura, Siew-Kee Low. Next-generation sequencing of circulating tumor DNA to monitor treatment response to CDK4/6 inhibitors in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A29.

MSK-ACCESS for the detection of fibroblast growth factor receptor-3 (FGFR3) mutations in plasma cell-free (cf)DNA of metastatic urothelial carcinoma (mUC) patients (pts) pre- and on erdafitinib (erda) therapy.

e17034 Background: The pan-FGFR inhibitor erda was recently FDA-approved for pretreated mUC pts harboring FGFR2/3 alterations. We explored concordance of FGFR3 mutation profiles between the primary tumor and cfDNA using the MSK-ACCESS platform. We also correlated changes in FGFR3 cfDNA mutant allele fraction (MAF) with response to drug. Methods: Plasma samples were collected from mUC pts started on erda at baseline, on treatment (tx), and at progression. Demographic and clinical characteristics were obtained. Baseline tumors were sequenced with MSK-IMPACT (ref) and plasma samples were sequenced using MSK-ACCESS, a cfDNA platform that sequences 129 genes using unique molecular indexes to generate > 15,000x coverage and detection of somatic mutations down to 0.1% MAF. Results: Between 08/2019-1/2020, 11 pts received erda 8mg daily. Of these, 3 pts increased dose to 9mg daily based on phosphorus level, 4 required dose reductions and 6 dose interruptions. In 5 pts, erda was discontinued for disease progression. FGFR3 S249C was the most frequent alteration detected (64%) followed by Y373C (18%), R248C and S371C (both 9%). Pre-treatment plasma FGFR3 profiles were concordant with tissue in 91% (10/11) of pts and additional FGFR3 mutations were detected in 3 cases (27%, Table), including 1 pt with an FGFR3-TACC3 fusion and hotspot mutations only in cfDNA. In 2 responding pts, the mutant allele was undetectable on erda. Conclusions: A high degree of concordance between primary tumor and cfDNA FGFR3 mutation detection was observed. FGFR3 mutations exclusive to cfDNA were found in a subset of pts. Further pt accrual and follow-up are ongoing to assess for correlations between erda response and tx-related changes in cfDNA MAF, and to assess whether cfDNA can identify resistance mechanisms. [Table: see text]

Analysis of BCR-IgH from genomic DNA with UMI incorporation

Abstract NGS of T and B cell immune receptors has become routine in immunological studies. While using RNA as a template has its benefits, gDNA is often preferred by those collecting the sample as DNA is often easier to obtain, especially from difficult samples or samples that have been in long-term storage. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may reflect the quantity of the repertoire better. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. Here, we describe the development of a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted uniquely since the relationship of one V(D)J per cell will be maintained. Thus, both the identity and the frequency of the B-cell can be captured directly, while allowing for the elimination of PCR and sequencing error. The newly developed panel was tested on a BCR-IgH Synthetic Library. The results demonstrate a high degree of correlation with the known BCR-IgH Synthetic Library (R20.956) including coverage of all known V and J gene targets. Currently, gDNA extracted from B-cell enriched PBMC samples are being tested. This method has important implications for monitoring aberrant clonotypes in blood cancers since the frequency of a clonotype can be directly quantified, even under conditions when one clonotype is expanded.

Robust T- and B-cell clonotype detection using simultaneous targeted mRNA and immune repertoire profiling of single cells

Abstract The key cell types contributing to the diversity of the immune repertoire are the T cells, with their T-cell receptor (TCR) and B cells with their B-cell receptor (BCR). During T- and B-cell development, the cell undergoes recombination of the germline gene segments, variable (V), diversity (D), and joining (J), also called V(D)J recombination. We developed a single cell clonotype detection assay for the BD Rhapsody™ Single-Cell Analysis System capable of processing thousands of single cells at once. We aimed to capture ~20,000 peripheral blood mononuclear cells from each individual donor (total of five) in a BD Rhapsody™ cartridge and took a sub-set of ~3,000 cells for immune repertoire profiling analysis of the complementarity determining region 3 (CDR3) of the variable regions of TCRα/TCRβ and the IgG heavy/light chains. The BD Rhapsody® Human Immune Response Panel for targeted mRNA gene expression analysis was used to classify the lymphocytes and monocyte populations. Results demonstrate that the BD Rhapsody™ VDJ assay can detect clonotype expression differences among different donors with >80% sensitivity and specificity. The abundance of TCR and BCR transcripts was determined by the addition of a unique molecular index to each mRNA molecule. Overall, the current technology demonstrates the ability to evaluate immune populations using single cell next generation sequencing. This assay could be applied not only to study the immune repertoire but to understand the biology of immune-related diseases and immune response to therapies. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Abstract P5-01-14: The combined blood analysis of cell-free DNA and genomic DNA of circulating tumor cells reveals additive value in hormone receptor-positive, HER2-negative metastatic breast cancer patients

Abstract Background: To gain comprehensive insights into the genomic complexity useful for therapy management in metastatic breast cancer (MBC), we aimed to isolate and analyze genomic DNA (gDNA) from circulating tumor cells (CTCs) and matched cell-free DNA (cfDNA) from a minimized blood volume. Methods: EDTA blood was drawn from 27 MBC patients with hormone receptor-positive and HER2 negative primary tumors at the time of disease progression. CTCs and CTC mRNA were isolated from 2 × 5 ml whole blood using the AdnaTest EMT2/StemCell Select/Detect. Plasma of CTC-depleted blood was used for cfDNA isolation. gDNA from CTCs was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/RNA Nano Kit prototype. CTC gDNA and cfDNA were analyzed with a customized QIAseq Targeted DNA Panel for Illumina with unique molecular indices (UMIs) analyzing AKT1, AR, BRCA1, BRCA2, EGFR, ERCC4, ERBB2, ERBB3, ESR1, FGFR1, KRAS, MUC16, PIK3CA, PIK3R1, PTEN, PTGFR and TGFB1. The library preparation protocol was slightly modified for the usage of CTC gDNA using the entire amount of the CTC gDNA eluate as input without pre-quantification followed by fragmentation and ligation with an increased number of adapters. All libraries were analyzed by paired-end sequencing on the Illumina NextSeq Sequencer with a NextSeq 550 System High-Output Kit, 2 × 150 bp reads with a mean coverage of 11000 x (CTC gDNA) and 8000 x (cfDNA). Consumables: QIAGEN, Germany. Results: Isolation of CTC gDNA and cfDNA was successfully established in a condensed workflow. The UMI coverage observed for cfDNA (2000 x) differed from the UMI coverage for CTC gDNA (500 x), resulting in dramatically increased lowest detectable allele frequency (AF) called with a confidence of 90% in CTC gDNA compared to cfDNA. On average, 6 CTC gDNA variants and 2 cfDNA variants were detected in each patient. Most variants were found in the MUC16 gene in both analytes. BRCA2 variants were the second most prevalent variants in CTC gDNA and cfDNA. PIK3CA and ESR1 variants were less common in CTC gDNA compared with cfDNA, while ERBB2 variants were only detected in CTC gDNA. 57% of all cfDNA variants (29/51) were recovered in the matched CTC gDNA, while 89% of all variants were unique in either CTC gDNA (125 variants) or cfDNA (22 variants). On average, the cfDNA variants with low AF (mean 15%) were not detected in CTC gDNA, while the cfDNA variants that were also found in CTC gDNA exhibited a mean AF of 44%. Similarly, the unique CTC gDNA variants had a mean AF of 23%, while the shared variants were prevalent with a mean AF of 47% in the CTC gDNA fraction. Comparing the patients, 11%/14% of the single CTC gDNA / cfDNA variants were detected in more than one patient, hence the majority of variants was patient-specific. The portion of patients without detectable cfDNA variants or CTC gDNA variants was 15% (4/27) / 11% (3/27), but combined analysis of CTC gDNA and cfDNA identified variants in 26 of all 27 MBC patients (96%). Conclusion: cfDNA and CTC gDNA showed additive variant information. Thus, it is advised to assess cfDNA and CTC gDNA variants to receive a comprehensive genomic picture that might enable to identify the most suitable therapy regimen in each individual patient in the future. Citation Format: Corinna Keup, Ann-Kathrin Bittner, Oliver Hoffmann, Mitra Tewes, Markus Storbeck, Peter Hahn, Siegfried Hauch, Markus Sprenger-Haussels, Rainer Kimmig, Sabine Kasimir-Bauer. The combined blood analysis of cell-free DNA and genomic DNA of circulating tumor cells reveals additive value in hormone receptor-positive, HER2-negative metastatic breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-14.

Abstract P4-10-27: Genomic aberrations in circulating tumor DNAs associated with progression in palbociclib-treated metastatic breast cancer patients

Abstract Background: Palbociclib is the first-in-class cyclin dependent kinase (CDK) 4/6 inhibitor approved for the treatment of patients (pts) with hormone receptor (HR) positive/human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer (MBC). Despite an initial response, pts eventually develop resistance to therapy; however, the underlying molecular mechanisms remain elusive. This study aimed to identify genomic aberrations in circulating tumor DNA (ctDNA) associated with treatment resistance and disease progression in MBC pts receiving palbociclib. Methods: Blood samples were collected from pts with HR+/HER2− MBC who were treated with palbociclib plus endocrine therapies (letrozole or fulvestrant). Circulating cell-free DNAs were isolated from plasma and assayed by targeted-capture next-generation sequencing using an in-house developed panel that covers the exons in 92 breast cancer-related genes. Unique molecular index (UMI)-based sequencing was performed using the Illumina HiSeq 4000 system. Progression free survival (PFS) differences between pts with different mutation status were compared using the log-rank test. The Cox proportional hazards model with time-dependent covariates was used to evaluate associations between mutations and PFS. Results: The study included 16 pts with a median age of 54.7 years (range: 32.6-74.8 years). A total of 42 blood samples were collected, including 16 baseline samples before the initiation of palbociclib +/- endocrine therapies (within 1.5 months prior to initiation), 10 samples during treatment (1.0-3.9 months after initiation), and 16 after treatment completion (within 1.2 months after completion). During a median follow-up of 23.7 weeks (range: 6.0-99.6 weeks), 8 pts had disease progression at initial blood collection after treatment initiation, and 11 had disease progression at final blood collection during follow-up. Among the 3 pts without progression, 2 were lost to follow-up and 1 had a change in regimen due to adverse events. Mutations were detected in 77 out of 92 genes. Mutations were identified for PIK3CA, ALK, ERBB2, NOTCH1, IDH1 in the baseline samples of 7, 3, 7, 6, and 12 patients, respectively. A shorter time to progression was observed in the pts with PIK3CA, ALK, ERBB2, or NOTCH1 mutations (log-rank P = 0.011, 0.016, 0.057, and 0.059, respectively), whereas those pts with IDH1 mutations had a prolonged PFS (log-rank P = 0.005). Similar results were obtained when the events and survival time at the final blood collection were used for analyses (log-rank P = 0.007, 0.004, 0.046, 0.023, and 0.001 for PIK3CA, ALK, ERBB2, NOTCH1, and IDH1 mutations, respectively). To analyze the association between a given mutation and disease progression, we separated pts into four risk groups according to the status change of the mutation prior to and after treatment: Group 1, persistent absence of the mutation; Group2, loss of the mutation after treatment; Group 3, acquisition of the mutation after treatment; Group 4, persistent presence of the mutation. Longitudinal analyses showed that PIK3CA E542K, ERBB2 P1035H, and NOTCH1 K1069Q mutations were associated with unfavorable survival. For PIK3CA E542K mutation, pts in Group 2 had an increased risk for progression, as compared with those in Group 1 (hazard ratio [HR] =9.90, P = 0.110). For ERBB2 P1035H and NOTCH1 K1069Q mutations, when compared with pts in Group 1, those in Group 4 had the highest risk of progression (HR = 17.24, P = 0.049; HR = 19.17, P = 0.043, respectively), whereas pts in Group 2 had a non-significant slightly increased risk. Conclusions: Results of this study show that genomic aberrations in ctDNA were associated with progression in MBC pts receiving palbociclib-containing regimens. Further research is needed to validate these findings in larger independent studies. Citation Format: Maysa M Abu-Khalaf, Chun Wang, Zhong Ye, Zhenchao Zhang, Daniel Silver, Frederick Fellin, Saveri Bhattacharya, Rebecca Jaslow, Terrence Cescon, AnaMaria Lopez, Ronald Myers, Qiang Wei, Bingshan Li, Hushan Yang. Genomic aberrations in circulating tumor DNAs associated with progression in palbociclib-treated metastatic breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-27.

Detection of Multiple Recurrent Novel BCL2 Mutations Co-Occurring with BCL2 Gly101Val in Patients with Chronic Lymphocytic Leukemia on Long Term Venetoclax

The BCL2 Gly101Val mutation may be acquired in patients with chronic lymphocytic leukaemia (CLL) treated with venetoclax (VEN), leading to reduced drug binding affinity and secondary resistance. In the majority of patients, the Gly101Val mutation is subclonal within the CLL compartment consistent with the presence of alternative resistance mechanisms in CLL cells not harboring the Gly101Val mutation. To date, two Gly101Val mutated patients have been identified with co-existing candidate resistance mechanisms in Gly101Val non-mutated cells; one with BCL-XL over-expression (Blombery et al, Cancer Discov., 2019) and another with a second subclonal candidate BCL2 resistance mutation - Asp103Tyr (Tausch et al, Haematologica 2019). Given the possibility of additional resistance mechanisms, we investigated patients with progressive CLL on VEN harboring the Gly101Val mutation for the presence of additional acquired resistance mutations in BCL2. Ten patients with progressive CLL on VEN with Gly101Val mutations were identified by sensitive allele-specific droplet digital PCR. To further assess for alternative BCL2 mutations in this cohort we performed ultra-deep amplicon-based next generation sequencing (NGS) (median depth ~50,000X) targeting BCL2. An amplicon variant caller (Canary) specifically designed for low level variant calling was used (Doig et al, BMC Bioinformatics, 2017). To achieve enhanced specificity we performed digital NGS with PCR error-correction using unique molecular indexes (UMI) (QiaSEQ Targeted DNA Panel). Given the high GC content of BCL2 we also used hybridization-based NGS using a custom targeted panel (Blombery et al, BJH 2017) combined with a sensitive unpaired variant caller (GATK4/Mutect2). In 7/10 (70%) patients, BCL2 mutations in addition to the Gly101Val were detected. Recurrent mutations (detected in more than one patient) were Asp103Tyr, Asp103Glu, Arg107_Arg110dup, and Val156Asp. All additional recurrent mutations were confirmed to be absent prior to commencing VEN (sensitivity 1% variant allele frequency[VAF]). Phase-analysis of NGS reads was consistent with the presence of the additional recurrent mutations on different alleles (and therefore cells, assuming heterozygosity) to both each other and to Gly101Val. Multiple addition recurrent mutations were observed in patients in the cohort with one patient harboring three recurrent mutations in addition to the Gly101Val (Asp103Tyr, Asp103Glu, Val156Asp). In multiple patients in the cohort, the VAF of non-Gly101Val mutations exceeded that of the Gly101Val mutation. Importantly, in all patients a significant (albeit variable) proportion of CLL cells were found to be BCL2 wild-type consistent with the presence of as yet unidentified resistance mechanisms unrelated to BCL2 mutations. In one patient, two additional non-recurrent mutations were observed (Ala113Gly and Arg129Leu) in addition to Gly101Val and Val156Asp. Again, all four mutations in this patient were observed to be in mutually exclusive NGS reads. Strikingly, all of the recurrent acquired BCL2 mutated residues identified in our cohort are situated in the BCL2 binding groove that binds VEN (Figure 1). The Asp103 codon in the P4 pocket is critical for VEN binding through hydrogen bonding between its sidechain and the azaindole moiety of VEN. The Asp103Glu mutation is noteworthy given that the equivalent residue to Asp103 in BCL-XL is a Glu, which reduces VEN binding to BCL-XL. The Val156 mutation situated at the base of the P2 pocket is close to the chlorophenyl moiety of VEN and a change to Asp in this position may disrupt VEN binding. Ongoing binding experiments and modeling in cellular systems will further elucidate the mechanism and contributions of these new recurrent mutations to VEN resistance. In summary, we have extended the landscape of acquired candidate resistance mutations occurring in patients treated with VEN to include four novel recurrent BCL2 mutations. Moreover, our data are consistent with the emerging observation of multiple acquired resistance mechanisms operating in different CLL cells in a single patient contributing to an "oligoclonal" pattern of clinical relapse on VEN therapy. Figure 1 - BCL2 protein structure surface bound to venetoclax (VEN) in orange. The Asp103Tyr, Asp103Glu and Val156Asp mutation sites are shown in red and Arg107_Arg110dup region in blue Disclosures Blombery: Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Seymour:Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; BeiGene: Research Funding.

Combining Gene Expression Profiling and Artificial Intelligence to Diagnose B-Cell Non-Hodgkin Lymphoma

Combining Gene Expression Profiling and Artificial Intelligence to Diagnose B-Cell Non-Hodgkin Lymphoma

Forensic analysis of MPS mtDNA data using QIAGEN biomedical genomics workbench and AQME tool – preliminary results

Massively Parallel Sequencing (MPS) allows to sequence the total mitochondrial DNA (mtDNA) through specific panels. Human Mitochondria Panel is one of the QIAseq Targeted DNA panels developed by QIAGEN for MPS, which have an optimized chemistry based on the integration of unique molecular indices (UMIs). However, the major problem is the analysis of the MPS data and, therefore, it became necessary to create bioinformatics tools that would enable mtDNA analysis such as the AQME tool to be used with the QIAGEN Biomedical Genomics Workbench software. In this study, a coverage comparison workflow was tested to verify the coverage differences between two mtDNA panels, QIAseq Human Mitochondria Panel and Precision ID mtDNA Whole Genome Panel. The preliminary results showed a significant decrease in the Precision ID coverage, after the bioinformatics removal of PCR artifacts. For the QIAseq panel, this coverage variation was lower, since the UMIs enable to know more accurately the initial number of molecules in the original mtDNA sequence.

150P - Liquid biopsy assays using combined circulating tumour cells and circulating tumour DNA in the same patients for the diagnosis of primary lung cancer

BackgroundLung cancer is the most common cause of cancer-related mortality worldwide. Early diagnosis and surgery may be one of the most important strategy for the treatment of lung cancer patients. However, the procedure of lung cancer diagnosis from initial suspicion to final confirmation is not efficient due to low sensitivity of current diagnostic methods and difficulties involved in tumor tissue biopsy in lung cancer compared to other cancer types. Therefore, we investigated the feasibility of liquid biopsy using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis for the better diagnosis of lung cancer. MethodsIn this study, we blindly analyzed both CTCs and ctDNA in the blood samples derived from 109 patients including histology-proven and clinically suspicious lung cancer patients to test the sensitivity of liquid biopsy methods. CTC analysis was done by CytoGen’s liquid biopsy platform, in which viable CTCs were isolated by size-based filtration with gravity and validated by immunostaining of EpCAM or CK, excluding CD45 positive cells and enumerated by CytoGen’s cell imaging software. Lung cancer diagnosis was predicted by a cut-off, 2 ≥ CTC in 5ml peripheral blood. For ctDNA analysis, EDGC F-Can platform was used to analyze single nucleotide variations of very low variant allele frequencies by utilizing unique molecular indexes and a novel read error correction algorithm. For comparison, we also analyzed the levels of conventional tumor markers (CEA, cyfra21-1 and NSE) in the patient cohort. ResultsCompared to the diagnostic sensitivities of conventional tumor markers (CEA 29%; cyfra21-1 41%; NSE 39%), both assays of CTCs and ctDNA showed higher diagnostic sensitivity in predicting primary lung cancer (CTCs 67%; ctDNA 83%). When the assays of CTCs and ctDNA were combined for the diagnosis, the sensitivity was increased up to 98%. ConclusionsCollectively, this study suggests that combined CTCs and ctDNA assay would be useful for the diagnosis of primary lung cancer. Legal entity responsible for the studyCytogen, Inc., EDGC, Inc., Department of Medicine, Samsung Medical Center FundingHas not received any funding DisclosureAll authors have declared no conflicts of interest.

Deep sequencing of B cell receptor repertoire

Immune repertoire is a collection of enormously diverse adaptive immune cells within an individual. As the repertoire shapes and represents immunological conditions, identification of clones and characterization of diversity are critical for understanding how to protect ourselves against various illness such as infectious diseases and cancers. Over the past several years, fast growing technologies for high throughput sequencing have facilitated rapid advancement of repertoire research, enabling us to observe the diversity of repertoire at an unprecedented level. Here, we focus on B cell receptor (BCR) repertoire and review approaches to B cell isolation and sequencing library construction. These experiments should be carefully designed according to BCR regions to be interrogated, such as heavy chain full length, complementarity determining regions, and isotypes. We also highlight preprocessing steps to remove sequencing and PCR errors with unique molecular index and bioinformatics techniques. Due to the nature of massive sequence variation in BCR, caution is warranted when interpreting repertoire diversity from error-prone sequencing data. Furthermore, we provide a summary of statistical frameworks and bioinformatics tools for clonal evolution and diversity. Finally, we discuss limitations of current BCR-seq technologies and future perspectives on advances in repertoire sequencing.

Abstract 2277: Workflow evaluation for molecular characterization of single circulating tumor cells in blood samples of patients with gynecological malignancies

Abstract Background: We recently presented the feasibility of single circulating tumor cell (CTC) analysis using the QIAscout single cell isolation platform (QIAGEN), followed by whole transcriptome amplification (WTA) and targeted Next Generation Sequencing (NGS). We here applied this workflow in blood samples of patients (pts) with metastatic breast cancer (MBC) and primary ovarian cancer (POC) and compared the result with a new workflow for single cell transcriptome analysis applying the QIAseq UPX 3' Transcriptome Kit (QIAGEN) for high throughput gene expression analysis. Methods: 5 ml blood of 7 MBC and 3 POC pts were analyzed for CTCs using the AdnaTest EMT2/StemCell SelectTM, followed single cell isolation. Adhesion of CTCs to the QIAscout array microrafts was supported by pre-treating the array with Corning® Cell-TakTM Adhesive. Single CTCs have been identified utilizing an inverted microscope, and isolated with the QIAscout single cell isolation platform. Subsequently, mRNA of single cells was amplified by WTA (REPLI-g WTA Single Cell Kit, QIAGEN) and cDNA of the cells was further analyzed by targeted NGS using a QIAseq™ Targeted RNA Panel (Human Cancer Transcriptome Panel, 395 genes). Applying the QIAseq UPX 3' Transcriptome Kit, each single cell was tagged with an individual cell ID-sequence and each RNA molecule was simultaneously tagged with an unique molecular index (UMI) during reverse transcription. Tagging of each individual cDNA molecule enabled pooling of all samples for subsequent amplification and library preparation steps, followed by high-throughput NGS. Methods were validated in spike-in experiments using different cell lines spiked into blood of healthy donors. Results: Using the QIAscout in combination with the WTA-workflow, we have already demonstrated an absent leukocyte contamination. However, only a very few of the detected cells in MBC pts (15%) were finally applicable for NGS. Nevertheless, analyzable CTCs expressed genes involved in angiogenesis (ITGB3), cell cycle (CCND2) and apoptosis (FASLG & SKP2). Applying the UPX 3' Transcriptome-workflow, the number of analyzable CTCs could be markedly improved as documented in spike-in experiments as well as in blood samples of MBC and POC pts. Up to now, in the latter group, we were able to isolate a total number of 84 CTCs [median 25 CTCs (range 24-35 cells)] with 44/84 (52%) ID-sequences detected, being applicable for NGS. Experiments and detailed transcriptome profiling of CTCs are ongoing to be presented at the meeting. Conclusion: Although both described workflows have to be further evaluated in more detail, the QIAscout single cell isolation platform allows subsequent transcriptome analysis followed by NGS to get insights into single cell heterogeneity for further therapeutic strategies. Citation Format: Janina Levermann, Norbert Hochstein, Paul Buderath, Benjamin Franken, Siegfried Hauch, Charline Bemmann, Rainer Kimmig, Sabine Kasimir-Bauer. Workflow evaluation for molecular characterization of single circulating tumor cells in blood samples of patients with gynecological malignancies [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 2277.

Abstract 1369: Establishment of a workflow for analysis of mRNA and gDNA from circulating tumor cells, extracellular vesicles and cell-free DNA from the same blood sample to mirror the genomic and transcriptomic complexity in metastatic breast cancer patients

Abstract Background: Blood analytes derived from liquid biopsies are discussed as useful tools for therapy stratification and for monitoring of clonal evolution. To gain comprehensive insights into the genomic and transcriptomic complexity in metastatic breast cancer (MBC) useful for therapy management, we aimed to isolate and analyze mRNA and gDNA from circulating tumor cells (CTCs), mRNA from extracellular vesicles (EVs) and cell-free DNA (cfDNA) from the same blood sample with minimized volume in a condensed workflow. Patients and Methods: EDTA blood (2x 9 ml) was drawn from 35 MBC patients with hormone receptor positive and HER2 negative primary tumor at time of disease progression and at two further consecutive staging time points. CTCs were isolated in duplicate from 5ml blood by immunomagnetic selection (AdnaTest EMT2/StemCell Select, QIAGEN). Plasma (4 ml) of the CTC-depleted blood was used for cfDNA isolation (QIAamp MinElute ccfDNA Kit, QIAGEN), plasma (4 ml) from the blood not used for CTC/cfDNA isolation was applied for EV isolation (exoRNeasy, QIAGEN). The mRNA purified from CTCs and EVs was analyzed by qPCR panel (AdnaPanel TNBC prototype, QIAGEN), while cfDNA was analyzed with a customized QIAseq Targeted DNA Panel for Illumina (QIAGEN) with unique molecular indices. We are working on a workflow to isolate gDNA from CTCs, starting from mRNA-depleted CTC lysates and analyzing the gDNA with a customized QIAseq Targeted DNA Panel. Isolation and mutation analysis of CTC gDNA was shown to be feasible in spiking experiments. Results: Isolation of mRNA and gDNA from CTCs, mRNA from EVs and cfDNA was successfully established in a parallel workflow. CTC and EV mRNA profiles showed substantial differences synergizing with regard to their clinical relevance. Whereas overexpression of mTOR was related to therapy responsiveness in CTCs, mTOR signals in EVs related to therapy failure. ERBB2 overexpressing CTCs were found in one third of all MBC patients enabling new therapeutic options. Matched cfDNA revealed the appearance of pathogenic mutations (e.g. PIK3CA H1047R) across treatment indicating underlying resistance mechanisms. Moreover, we identified significant correlations of therapy outcome with the overexpression of transcripts/ presence of mutations in each of the isolated liquid biopsy analytes. Comparison of CTC gDNA and cfDNA is further conducted. Conclusions: We were able to describe a complete workflow for parallel CTC mRNA, CTC gDNA, EV mRNA and cfDNA isolation from a minimized blood volume. In this research study each analyte showed synergistic potential for therapy management, thus the comprehensive picture of the genomic and transcriptomic complexity might in future enable to identify the most suitable therapy regiment in each individual patient. Citation Format: Corinna Keup, Markus Storbeck, Peter Hahn, Siegfried Hauch, Markus Sprenger-Haussels, Ann-Kathrin Bittner, Oliver Hoffmann, Mitra Tewes, Rainer Kimmig, Sabine Kasimir-Bauer. Establishment of a workflow for analysis of mRNA and gDNA from circulating tumor cells, extracellular vesicles and cell-free DNA from the same blood sample to mirror the genomic and transcriptomic complexity in metastatic breast cancer patients [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 1369.

Abstract 2477: CopyCat: estimating genomic copy number profiles from high-throughput single cell RNA seq data

Abstract CopyCat is a computational approach for estimate genomic-copy number profiles from high-throughput single cell RNAseq data that is generated by technologies, such as Drop-Seq, 10X genomics or Nanowells. CopyCat involves first normalizing and transforming unique molecular index (UMI) count data matrices to calculate single cell copy number states from low-coverage datasets using gene-bin windows across the human genome, and does not require a reference normal cell control. The resulting binned data is normalized and used for segmentation to reduce noise from individual genes that can contribute to gene dosage effects along chromosomes. We show that this methods is highly effective at distinguishing diploid and aneuploid cells in human tumors or cancer cell lines, and can efficiently detect major (>10mb) chromosomal amplifications and deletions compared to copy number data generated by DNA sequencing of match samples. We further show that this method can resolve some clonal substructure within individual tumors, when subpopulations are distinguished by large chromosomal events. Our method outputs segmented copy number profiles and predicted classifications of normal (diploid) and tumor (aneuploid) cell states for individual single cells. It is implemented in R and is available on Github. Citation Format: Ruli Gao, Nicholas Navin. CopyCat: estimating genomic copy number profiles from high-throughput single cell RNA seq data [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 2477.

Abstract 1346: Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay

Abstract Checkpoint inhibitor (CPI) therapy demonstrates a remarkable clinical benefit in many cancer types. However, the ability to successfully select patients who will benefit from CPIs is still limited. Tumor mutational burden (TMB), a measure of the number of somatic mutations per coding area of tumor genome, is a putative biomarker of response showing great promise in CPI and immunotherapy combination trials. The ability to measure TMB from tumor biopsies or plasma samples will be important for clinical adoption of this biomarker. Herein we report on initial performance evaluation of Illumina’s TruSight™ Oncology 500 gene (TSO500) NGS assay for the analysis of TMB in FFPE tissue and plasma cell-free (cf)DNA specimens. Illumina’s TSO500 assay employs hybrid-capture based approach for target enrichment coupled with unique molecular indices to enable low frequency variant detection of single nucleotide variants and indels. This comprehensive cancer panel interrogates relevant cancer biomarkers in >500 cancer genes (~2 Mb) from as little as 40 ng of FFPE DNA or 30 ng of cfDNA. In addition to variant calls, Illumina’s analysis pipeline reports a TMB score and microsatellite instability (MSI) status. Results obtained with the TSO500 TMB assay were compared to our validated whole exome sequencing (WES) TMB assessment for FFPE tissue specimens, with and without matched normal. As previously reported, the WES TMB tumor-only pipeline uses somatic variant classifications determined using a random forest model to generate TMB scores from analysis of tumor FFPE specimens. TSO500 TMB and MSI statues were evaluated in a set of 50 tumor FFPE specimens obtained from late-stage (III+) colorectal and bladder cancer patients. TSO500 TMB results correlated well with the WES-derived TMB results, although some differences were observed in the magnitude of high TMB scores. TSO500 TMB levels correlated strongly with the MSI status, with 100% of MSI-high tumors demonstrating high TMB scores (> 20), and greater than 85% of MSI-low tumors characterized as TMB-low (<20%). Additionally, correlations between tissue and blood-based TMB assessments in matched FFPE and plasma specimens are reported. Overall, our results demonstrate a strong correlation between TSO500 panel-based and whole exome sequencing-based TMB results in tissue and report comparisons to TMB calling in liquid biopsies. Our data further confirm that tumor mutational load is strongly connected to DNA damage repair deficiency in colorectal and bladder cancer patients. Citation Format: Stephanie B. Hastings, Gunjan Hariani, Victor J. Weigman, Tingting Jiang, Chen Zhao, Rajiv Raja, Traci Pawlowski, Philip Brohawn, Patrick Hurban. Panel-based tumor mutational burden (TMB) analysis of matched tumor and plasma specimens using Illumina’s TruSight Oncology 500 next-generation sequencing assay [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 1346.

Targeted deep sequencing revealed variants in cell-free DNA of hormone receptor-positive metastatic breast cancer patients

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, AR, BRCA1, BRCA2, EGFR, ERCC4, ERBB2, ERBB3, ESR1, FGFR1, KRAS, MUC16, PIK3CA, PIK3R1, PTEN, PTGFR, and TGFB1. CfDNA was sequenced on the NextSeq® 550 platform (Illumina) and variants were analyzed with Ingenuity Variant Analysis (QIAGEN). We evaluated cfDNA variants in 40 of the 44 hormone receptor-positive and HER2-negative patients with a high mean coverage of 22,000×, resulting in MUC16, BRCA2, ERBB3, and AR variant calling in > 90% of the patients. 47% of all AR variants were pathogenic and at least one pathogenic or likely pathogenic variant was detected in each patient. A specific BRCA1 variant and > 3.5 pathogenic variants significantly associated with a reduced survival after diagnosis of metastasis. Longitudinal monitoring revealed an increase of pathogenic and likely pathogenic PIK3CA and ESR1 variant allele frequency under everolimus and exemestane, 8 months before proof of therapy failure by visual staging in one exemplary case. The identification of new variants with high prevalence, prognostic value, and dynamics under treatment by deep sequencing of cfDNA might empower sensitive monitoring and personalized therapeutic decisions.

A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma

BackgroundCirculating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues.ResultsDifferent RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2–3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation.ConclusionsSmall RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.

Cell-Free DNA Variant Sequencing Using CTC-Depleted Blood for Comprehensive Liquid Biopsy Testing in Metastatic Breast Cancer.

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube” format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube” format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies.