Surpassing sensitivity limits in liquid biopsy.

Attenuation of cell-free DNA clearance in vivo is an alternative strategy to maximize recovery.

Bridging biological cfDNA features and machine learning approaches.

Liquid biopsies (LBs), particularly using circulating tumor DNA (ctDNA), are expected to revolutionize precision oncology and blood-based cancer screening. Recent technological improvements, in combination with the ever-growing understanding of cell-free DNA (cfDNA) biology, are enabling the detection of tumor-specific changes with extremely high resolution and new analysis concepts beyond genetic alterations, including methylomics, fragmentomics, and nucleosomics. The interrogation of a large number of markers and the high complexity of data render traditional correlation methods insufficient. In this regard, machine learning (ML) algorithms are increasingly being used to decipher disease- and tissue-specific signals from cfDNA. Here, we review recent insights into biological ctDNA features and how these are incorporated into sophisticated ML applications.

Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents.

Background: Treatment stratification and response assessment in pediatric sarcomas has relied on imaging studies and surgical/histopathological evidence of vital tumor cells. Such studies and evidence collection processes often involve radiation and/or general anesthesia in children. Cell-free circulating tumor DNA (ctDNA) detection in blood plasma is one available method of so-called liquid biopsies that has been shown to correlate qualitatively and quantitatively with the existence of vital tumor cells in the body. Our clinical observational study focused on the utility and feasibility of ctDNA detection in pediatric Ewing sarcoma (EWS) as a marker of minimal residual disease (MRD). Patients and methods: We performed whole genome sequencing (WGS) to identify the exact breakpoints in tumors known to carry the EWS-FLI1 fusion gene. Patient-specific fusion breakpoints were tracked in peripheral blood plasma using digital droplet PCR (ddPCR) before, during, and after therapy in six children and young adults with EWS. Presence and levels of fusion breakpoints were correlated with clinical disease courses. Results: We show that the detection of ctDNA in the peripheral blood of EWS patients (i) is feasible in the clinical routine and (ii) allows for the longitudinal real-time monitoring of MRD activity in children and young adults. Although changing ctDNA levels correlated well with clinical outcome within patients, between patients, a high variability was observed (inter-individually). Conclusion: ctDNA detection by ddPCR is a highly sensitive, specific, feasible, and highly accurate method that can be applied in EWS for follow-up assessments as an additional surrogate parameter for clinical MRD monitoring and, potentially, also for treatment stratification in the near future.

Molecular profiling of soft-tissue sarcomas with FoundationOne® Heme identifies potential targets for sarcoma therapy: a single-centre experience.

BACKGROUND: Molecular diagnosis has become an established tool in the characterisation of adult soft-tissue sarcomas (STS). FoundationOne® Heme analyses somatic gene alterations in sarcomas via DNA and RNA-hotspot sequencing of tumour-associated genes. METHODS: We evaluated FoundationOne® Heme testing in 81 localised STS including 35 translocation-associated and 46 complex-karyotyped cases from a single institution. RESULTS: Although FoundationOne® Heme achieved broad patient coverage and identified at least five genetic alterations in each sample, the sensitivity for fusion detection was rather low, at 42.4%. Nevertheless, potential targets for STS treatment were detected using the FoundationOne® Heme assay: complex-karyotyped sarcomas frequently displayed copy-number alterations of common tumour-suppressor genes, particularly deletions in TP53, NF1, ATRX, and CDKN2A. A subset of myxofibrosarcomas (MFS) was amplified for HGF (n = 3) and MET (n = 1). PIK3CA was mutated in 7/15 cases of myxoid liposarcoma (MLS; 46.7%). Epigenetic regulators (e.g. MLL2 and MLL3) were frequently mutated. CONCLUSIONS: In summary, FoundationOne® Heme detected a broad range of genetic alterations and potential therapeutic targets in STS (e.g. HGF/MET in a subset of MFS, or PIK3CA in MLS). The assay's sensitivity for fusion detection was low in our sample and needs to be re-evaluated in a larger cohort.

Somatic Copy-Number Alterations in Plasma Circulating Tumor DNA from Advanced EGFR-Mutated Lung Adenocarcinoma Patients.

BACKGROUND: To assess the clinical relevance of genome-wide somatic copy-number alterations (SCNAs) in plasma circulating tumor DNA (ctDNA) from advanced epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma patients. METHODS: We included 43 patients with advanced EGFR T790M-positive lung adenocarcinoma who were treated with osimertinib after progression under previous EGFR-TKI therapy. We performed genomic profiling of ctDNA in plasma samples from each patient obtained pre-osimertinib and after patients developed resistance to osimertinib. SCNAs were detected by shallow whole-genome plasma sequencing and EGFR mutations were assessed by droplet digital PCR. RESULTS: SCNAs in resistance-related genes (rrSCNAs) were detected in 10 out of 31 (32%) evaluable patients before start of osimertinib. The presence of rrSCNAs in plasma before the initiation of osimertinib therapy was associated with a lower response rate to osimertinib (50% versus 81%, p = 0.08) and was an independent predictor for shorter progression-free survival (adjusted HR 3.33, 95% CI 1.37-8.10, p = 0.008) and overall survival (adjusted HR 2.54, 95% CI 1.09-5.92, p = 0.03). CONCLUSIONS: Genomic profiling of plasma ctDNA is clinically relevant and affects the efficacy and clinical outcome of osimertinib. Our approach enables the comprehensive assessment of SCNAs in plasma samples of lung adenocarcinoma patients and may help to guide genotype-specific therapeutic strategies in the future.

Dynamic Changes of Circulating Tumor DNA Predict Clinical Outcome in Patients With Advanced Non-Small-Cell Lung Cancer Treated With Immune Checkpoint Inhibitors.

PURPOSE: Immune checkpoint inhibitors (ICIs) are increasingly being used in non-small-cell lung cancer (NSCLC), yet biomarkers predicting their benefit are lacking. We evaluated if on-treatment changes of circulating tumor DNA (ctDNA) from ICI start (t0) to after two cycles (t1) assessed with a commercial panel could identify patients with NSCLC who would benefit from ICI. PATIENTS AND METHODS: The molecular ctDNA response was evaluated as a predictor of radiographic tumor response and long-term survival benefit of ICI. To maximize the yield of ctDNA detection, de novo mutation calling was performed. Furthermore, the impact of clonal hematopoiesis (CH)-related variants as a source of biologic noise was investigated. RESULTS: After correction for CH-related variants, which were detected in 75 patients (44.9%), ctDNA was detected in 152 of 167 (91.0%) patients. We observed only a fair agreement of the molecular and radiographic response, which was even more impaired by the inclusion of CH-related variants. After exclusion of those, a ≥ 50% molecular response improved progression-free survival (10 v 2 months; hazard ratio [HR], 0.55; 95% CI, 0.39 to 0.77; P = .0011) and overall survival (18.4 v 5.9 months; HR, 0.44; 95% CI, 0.31 to 0.62; P < .0001) compared with patients not achieving this end point. After adjusting for clinical variables, ctDNA response and STK11/KEAP1 mutations (HR, 2.08; 95% CI, 1.4 to 3.0; P < .001) remained independent predictors for overall survival, irrespective of programmed death ligand-1 expression. A landmark survival analysis at 2 months (n = 129) provided similar results. CONCLUSION: On-treatment changes of ctDNA in plasma reveal predictive information for long-term clinical benefit in ICI-treated patients with NSCLC. A broader NSCLC patient coverage through de novo mutation calling and the use of a variant call set excluding CH-related variants improved the classification of molecular responders, but had no significant impact on survival.

A higher ctDNA fraction decreases survival in regorafenib-treated metastatic colorectal cancer patients. Results from the regorafenib's liquid biopsy translational biomarker phase II pilot study.

The predictive effect of circulating tumor DNA (ctDNA) in colorectal cancer (CRC) treatment is still highly discussed. The primary objective of our study was to investigate a possible prognostic/predictive value of ctDNA under regorafenib treatment. This prospective multicenter translational biomarker phase II pilot study enrolled 30 metastatic CRC patients (67% men, 33% women) treated with regorafenib. ctDNA was assessed in plasma before treatment start and at defined time points during administration. Measurement of tumor fraction as well as mutation and copy number analysis of CRC driver genes were performed by next-generation sequencing approaches. Multivariate analyses for survival and treatment efficacy were adjusted to age, gender and Eastern Cooperative Oncology Group. Disease control rate was 30%. Median tumor fraction at baseline was 18.5% (0-49.9). Mutations in CRC driver genes or genes involved in angiogenesis were identified in 25 patients (83.3%). KRAS mutations were detected in 13 of 14 KRAS-positive tumors; in three patients without KRAS mutation in the respective tumors, acquired mutations as a consequence of prior anti-EGFR treatment were detected. In a subset of patients, novel occurring mutations or focal amplifications were detected. A tumor fraction of 5% and higher at baseline was significantly associated with a decreased OS (P = .022; hazard ratio 3.110 (95% confidence interval: 1.2-8.2). ctDNA is detectable in a high proportion of mCRC patients. Higher ctDNA levels are associated with survival among regorafenib treatment. Moreover, our data highlight the benefit of a combined evaluation of mutations and somatic copy number alterations in advanced cancer patients.

On-treatment measurements of circulating tumor DNA during FOLFOX therapy in patients with colorectal cancer.

We addressed a significant unknown feature of circulating tumor DNA (ctDNA), i.e., how ctDNA levels change during chemotherapy, by serially monitoring ctDNA in patients with colorectal cancer during the 48-h application of FOLFOX. Surprisingly, we did not observe a spike in ctDNA as a sign of a responsive tumor, but instead ctDNA levels initially decreased and remained low in patients with stable disease or partial response. Our observations reveal further insights into cell destruction during chemotherapy with important implications for the management of patients.

A Multi-Analyte Approach for Improved Sensitivity of Liquid Biopsies in Prostate Cancer.

Novel androgen receptor (AR) signaling inhibitors have improved the treatment of castration-resistant prostate cancer (CRPC). Nonetheless, the effect of these drugs is often time-limited and eventually most patients become resistant due to various AR alterations. Although liquid biopsy approaches are powerful tools for early detection of such therapy resistances, most assays investigate only a single resistance mechanism. In combination with the typically low abundance of circulating biomarkers, liquid biopsy assays are therefore informative only in a subset of patients. In this pilot study, we aimed to increase overall sensitivity for tumor-related information by combining three liquid biopsy approaches into a multi-analyte approach. In a cohort of 19 CRPC patients, we (1) enumerated and characterized circulating tumor cells (CTCs) by mRNA-based in situ padlock probe analysis, (2) used RT-qPCR to detect cancer-associated transcripts (e.g., AR and AR-splice variant 7) in lysed whole blood, and (3) conducted shallow whole-genome plasma sequencing to detect AR amplification. Although 44-53% of patient samples were informative for each assay, a combination of all three approaches led to improved diagnostic sensitivity, providing tumor-related information in 89% of patients. Additionally, distinct resistance mechanisms co-occurred in two patients, further reinforcing the implementation of multi-analyte liquid biopsy approaches.

Publisher Correction: Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors.

BACKGROUND: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established. METHODS: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine. RESULTS: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings. CONCLUSIONS: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

Deregulation of transcription factors (TFs) is an important driver of tumorigenesis, but non-invasive assays for assessing transcription factor activity are lacking. Here we develop and validate a minimally invasive method for assessing TF activity based on cell-free DNA sequencing and nucleosome footprint analysis. We analyze whole genome sequencing data for >1,000 cell-free DNA samples from cancer patients and healthy controls using a bioinformatics pipeline developed by us that infers accessibility of TF binding sites from cell-free DNA fragmentation patterns. We observe patient-specific as well as tumor-specific patterns, including accurate prediction of tumor subtypes in prostate cancer, with important clinical implications for the management of patients. Furthermore, we show that cell-free DNA TF profiling is capable of detection of early-stage colorectal carcinomas. Our approach for mapping tumor-specific transcription factor binding in vivo based on blood samples makes a key part of the noncoding genome amenable to clinical analysis.

The transcription factor VvWRKY33 is involved in the regulation of grapevine (Vitis vinifera) defense against the oomycete pathogen Plasmopara viticola.

Grapevine (Vitis vinifera ssp. vinifera) is one of the most important fruit species; however, it is highly susceptible to various pathogens, which can cause severe crop losses in viticulture. It has been shown that several WRKY class transcription factors (TFs) are part of the signal transduction cascade, which leads to the activation of plant defense reactions against various pathogens. In the present investigation, a full-length cDNA was isolated from V. vinifera leaf tissue encoding a predicted protein, designated VvWRKY33, which shows the characteristics of group I WRKY protein family. VvWRKY33 induction correlates with the expression of VvPR10.1 (pathogenesis-related 10.1) gene in the leaves of the resistant cultivar 'Regent' after infection with Plasmopara viticola, whereas in the susceptible cultivar 'Lemberger' VvWRKY33 and VvPR10.1 are not induced. Corresponding expression of the TF and VvPR10.1 was even obtained in uninfected ripening berries. In planta, analysis of VvWRKY33 has been performed by ectopic expression of VvWRKY33 in grapevine leaves of greenhouse plants mediated via Agrobacterium tumefaciens transformation. In consequence, VvWRKY33 strongly increases resistance to P. viticola in the susceptible cultivar 'Shiraz' and reduces pathogen sporulation of about 50-70%, indicating a functional role for resistance in grapevine. Complementation of the resistance-deficient Arabidopsis thaliana Columbia-0 (Col-0) mutant line wrky33-1 by constitutive expression of VvWRKY33 restores resistance against Botrytis cinerea to wild-type level and in some complemented mutant lines even exceeds the resistance level of the parental line Col-0. Our results support the involvement of VvWRKY33 in the defense reaction of grapevine against different pathogens.