Targeted kinase inhibitors improve the prognosis of lung cancer patients with ALK alterations (ALK+). However, due to the emergence of acquired resistance and varied clinical trajectories, early detection of disease progression is warranted to guide patient management and therapy decisions. We utilized 343 longitudinal plasma DNA samples from 43 ALK+ NSCLC patients receiving ALK-directed therapies to determine molecular progression based on matched panel-based targeted next-generation sequencing (tNGS), and shallow whole-genome sequencing (sWGS). ALK-related alterations were detected in 22 out of 43 (51%) patients. Among 343 longitudinal plasma samples analyzed, 174 (51%) were ctDNA-positive. ALK variant and fusion kinetics generally reflected the disease course. Evidence for early molecular progression was observed in 19 patients (44%). Detection of ctDNA at therapy baseline indicated shorter times to progression compared to cases without mutations at baseline. In patients who succumbed to the disease, ctDNA levels were highly elevated towards the end of life. Our results demonstrate the potential utility of these NGS assays in the clinical management of ALK+ NSCLC.

Background: DNA methylation (5-mC) signals in cell-free DNA (cfDNA) of cancer patients represent promising biomarkers for minimally invasive tumor detection. The high abundance of cancer-associated 5-mC alterations permits parallel and highly sensitive assessment of multiple 5-mC biomarkers. Here, we performed genome-wide 5-mC profiling in the plasma of metastaticALK-rearranged non-small cell lung cancer (NSCLC) patients receiving tyrosine kinase inhibitor therapy. We established a strategy to identifyALK-specific 5-mC changes from cfDNA and demonstrated the suitability of the identified markers for cancer detection, prognosis, and therapy monitoring.Methods: Longitudinal plasma samples (n = 79) of 21ALK-positive NSCLC patients and 13 healthy donors were collected alongside 15ALK-positive tumor tissue and 10 healthy lung tissue specimens. All plasma and tissue samples were analyzed by cell-free DNA methylation immunoprecipitation sequencing to generate genome-wide 5-mC profiles. Information on genomic alterations (i.e., somatic mutations/fusions and copy number alterations) determined in matched plasma samples was available from previous studies.Results: We devised a strategy that identified tumor-specific 5-mC biomarkers by reducing 5-mC background signals derived from hematopoietic cells. This was followed by differential methylation analysis (cases vs. controls) and biomarker validation using 5-mC profiles ofALK-positive tumor tissues. The resulting 245 differentially methylated regions were enriched for lung adenocarcinoma-specific 5-mC patterns in TCGA data and indicated transcriptional repression of several genes described to be silenced in NSCLC (e.g.,PCDH10,TBX2,CDO1, andHOXA9). Additionally, 5-mC-based tumor DNA (5-mC score) was highly correlated with other genomic alterations in cell-free DNA (Spearman,ρ > 0.6), while samples with high 5-mC scores showed significantly shorter overall survival (log-rankp = 0.025). Longitudinal 5-mC scores reflected radiologic disease assessments and were significantly elevated at disease progression compared to the therapy start (p = 0.0023). In 7 out of 8 instances, rising 5-mC scores preceded imaging-based evaluation of disease progression.Conclusion: We demonstrated a strategy to identify 5-mC biomarkers from the plasma of cancer patients and integrated them into a quantitative measure of cancer-associated 5-mC alterations. Using longitudinal plasma samples of ALK-positive NSCLC patients, we highlighted the suitability of cfDNA methylation for prognosis and therapy monitoring.

Current strategies for the clinical management of prostate cancer are inadequate for a precise risk stratification between indolent and aggressive tumors. Recently developed tissue-based molecular biomarkers have refined the risk assessment of the disease. The characterization of tissue biopsy components and subsequent identification of relevant tissue-based molecular alterations have the potential to improve the clinical decision making and patient outcomes. However, tissue biopsies are invasive and spatially restricted due to tumor heterogeneity. Therefore, there is an urgent need for complementary diagnostic and prognostic options. Liquid biopsy approaches are minimally invasive with potential utility for the early detection, risk stratification, and monitoring of tumors. In this review, we focus on tissue and liquid biopsy biomarkers for early diagnosis and risk stratification of prostate cancer, including modifications on the genomic, epigenomic, transcriptomic, and proteomic levels. High-risk molecular alterations combined with orthogonal clinical parameters can improve the identification of aggressive tumors and increase patient survival.

Background: Outcomes under anti-PD-(L)1 therapy have been variable in advanced non-small cell lung cancer (NSCLC) without reliable predictive biomarkers so far. Targeted next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) has demonstrated potential clinical utility to support clinical decisions, but requires prior tumor genetic profiling for proper interpretation, and wide adoption remains limited due to high costs. Methods: Tumor-agnostic low-coverage ctDNA whole genome sequencing (lcWGS) was used to longitudinally track genome-wide copy number variations (CNVs) and fragmentation features in advanced NSCLC patients (n = 118 samples from 49 patients) and healthy controls (n = 57). Tumor PD-L1 expression was available for comparison. Findings: Fragmentation features and CNVs were complementary indicators, whose combination significantly increased ctDNA detection compared to single-marker assessments (+ 20.3% compared to CNV analysis alone). Baseline fragment length alterations, but not CNVs, were significantly associated with subsequent progression-free survival (PFS; hazard ratio [HR] = 4.10, p = 6.58e-05) and could improve PFS predictions based on tumor PD-L1 expression alone (HR = 2.70, p = 0.019). Residual CNVs or aberrant fragmentation of ctDNA under ongoing therapy could stratify patients according to the subsequent response duration (median 5.8 vs. 47.0 months, p = 1.13e-06). The integrative analysis of ctDNA fragment characteristics at baseline, tumor PD-L1 expression, and residual ctDNA under ongoing treatment constituted the strongest independent predictor of PFS (p = 6.25e-05) and overall survival (p = 1.3e-03) in multivariable analyses along with other clinicopathologic variables. Interpretation: This study demonstrates the feasibility and potential clinical utility of lcWGS for the tumor-agnostic stratification and monitoring of advanced NSCLC under PD-(L)1 blockade based on CNV and fragmentomic profiling.

Local recurrence after radiotherapy is common in locally advanced head and neck cancer (HNC) patients. Re‐irradiation can improve local disease control, but disease progression remains frequent. Hence, predictive biomarkers are needed to adapt treatment intensity to the patient's individual risk. We quantified circulating tumor DNA (ctDNA) in sequential plasma samples and correlated ctDNA levels with disease outcome. Ninety four longitudinal plasma samples from 16 locally advanced HNC patients and 57 healthy donors were collected at re‐radiotherapy baseline, after 5 and 10 radiation fractions, at irradiation end, and at routine follow‐up visits. Plasma DNA was subjected to low coverage whole genome sequencing for copy number variation (CNV) profiling to quantify ctDNA burden. CNV‐based ctDNA burden was detected in 8/16 patients and 25/94 plasma samples. Ten additional ctDNA‐positive samples were identified by tracking patient‐specific CNVs found in earlier sequential plasma samples. ctDNA‐positivity after 5 and 10 radiation fractions (both: log‐rank, p = .050) as well as at the end of irradiation correlated with short progression‐free survival (log‐rank, p = .006). Moreover, a pronounced decrease of ctDNA toward re‐radiotherapy termination was associated with worse treatment outcome (log‐rank, p = .005). Dynamic ctDNA tracking in serial plasma beyond re‐radiotherapy reflected treatment response and imminent disease progression. In five patients, molecular progression was detected prior to tumor progression based on clinical imaging. Our findings emphasize that quantifying ctDNA during re‐radiotherapy may contribute to disease monitoring and personalization of adjuvant treatment, follow‐up intervals, and dose prescription.