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.

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.

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.

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.

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.

Advances in endocrine therapies for hormone receptor (HR)-positive/HER2-negative advanced breast cancer (ABC) continue to transform care and significantly improve patient outcomes. However, the integration of molecular and clinical risk stratification into guiding individualized treatment selection remains a key challenge. We therefore evaluated whether the integration of baseline circulating tumor DNA (ctDNA)-derived tumor metrics enhances risk stratification among patients receiving early lines of treatment of HR-positive ABC in a real-world multicenter cohort of patients in Austria. Patients with HR-positive/HER2-negative ABC treated at multiple Austrian centers were included. CtDNA was analyzed using a 77-gene panel (AVENIO ctDNA Expanded Kit). Tumor fraction (TFx) was estimated via two complementary approaches: untargeted aneuploidy assessment using mFAST-SeqS, and the highest variant allele frequency (hVAF) from the AVENIO assay. Somatic variants and single, binary, and three-level composite TFx metrics were assessed for their association with progression-free and overall survival (PFS, OS). We analyzed 225 ctDNA samples from 184 patients [128 before first-line (1L) and 76 before second-line (2L) treatment], including 40 paired samples. Overall TFx was low (median z-score 2.49; range -0.5-208.3), with higher levels in 2L, although the difference did not reach statistical significance (P = 0.058). In contrast, the hVAF was significantly higher in the 2L cohort (P = 0.007). Somatic variant burden was significantly increased in 2L (P < 0.001), with notably more frequent ESR1 mutations (26.7% versus 7.1% in 1L). Median PFS was 29.2 months in 1L and 6.0 months in 2L, while median OS was 57.3 months and 16.1 months, respectively. TP53 and ESR1 mutations, and all ctDNA-based metrics were significantly associated with PFS and OS, with a three-level composite ctDNA variable showing the highest prognostic discrimination. Our findings demonstrate that integrating baseline ctDNA-derived TFx metrics with established clinical variables significantly improves risk stratification in HR-positive/HER2-negative ABC.