GCparagon: evaluating and correcting GC biases in cell-free DNA at the fragment level.

Analyses of cell-free DNA (cfDNA) are increasingly being employed for various diagnostic and research applications. Many technologies aim to increase resolution, e.g. for detecting early-stage cancer or minimal residual disease. However, these efforts may be confounded by inherent base composition biases of cfDNA, specifically the over - and underrepresentation of guanine (G) and cytosine (C) sequences. Currently, there is no universally applicable tool to correct these effects on sequencing read-level data. Here, we present GCparagon, a two-stage algorithm for computing and correcting GC biases in cfDNA samples. In the initial step, length and GC base count parameters are determined. Here, our algorithm minimizes the inclusion of known problematic genomic regions, such as low-mappability regions, in its calculations. In the second step, GCparagon computes weights counterbalancing the distortion of cfDNA attributes (correction matrix). These fragment weights are added to a binary alignment map (BAM) file as alignment tags for individual reads. The GC correction matrix or the tagged BAM file can be used for downstream analyses. Parallel computing allows for a GC bias estimation below 1 min. We demonstrate that GCparagon vastly improves the analysis of regulatory regions, which frequently show specific GC composition patterns and will contribute to standardized cfDNA applications.

Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma.

Leukemic cutaneous T-cell lymphomas (L-CTCL) are lymphoproliferative disorders of skin-homing mature T-cells causing severe symptoms and high mortality through chronic inflammation, tissue destruction, and serious infections. Despite numerous genomic sequencing efforts, recurrent driver mutations have not been identified, but chromosomal losses and gains are frequent and dominant. We integrated genomic landscape analyses with innovative pharmacologic interference studies to identify key vulnerable nodes in L-CTCL. We detected copy number gains of loci containing the STAT3/5 oncogenes in 74% (n = 17/23) of L-CTCL, which correlated with the increased clonal T-cell count in the blood. Dual inhibition of STAT3/5 using small-molecule degraders and multi-kinase blockers abolished L-CTCL cell growth in vitro and ex vivo, whereby PAK kinase inhibition was specifically selective for L-CTCL patient cells carrying STAT3/5 gains. Importantly, the PAK inhibitor FRAx597 demonstrated encouraging anti-leukemic activity in vivo by inhibiting tumor growth and disease dissemination in intradermally xenografted mice. We conclude that STAT3/5 and PAK kinase interaction represents a new therapeutic node to be further explored in L-CTCL.

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.

Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Influence of Bitter Taste on Affective Facial Processing: An ERP Study.

Previous research showed that a bitter taste in the mouth is able to enhance hostile response tendencies to social rejection. The present event-related potential (ERP) study sought to investigate neuronal components of this effect. We presented 52 participants (39 women and 13 men; mean age = 23.3 years) with images of facial expressions signaling social rejection (angry, disgusted) or no rejection (happy, neural), whereas they either experienced a bitter aftertaste (bitter group [BG]: n = 26) or rinsed their mouth with water (control group [CG]: n = 26). The BG rated the aftertaste as extremely intense and disgusting and showed a decreased parietal P200 to all facial expressions, as well as a lowered parietal P300 to cues of nonrejection. The bitter intervention neither influenced the affective ratings for the images nor self-reported hostility. In conclusion, the ERP findings indicated that bitterness first reduced visual attention to social cues in general (P200) and then specifically to cues of nonrejection (P300). Bitterness was not associated with increased sensitivity to disgust/anger signaled by others neither on a neuronal nor self-report level.

Disgust evoked by strong wormwood bitterness influences the processing of visual food cues in women: An ERP study.

The perception of intense bitterness is associated with disgust and food rejection. The present cross-modal event-related potential (ERP) study investigated whether a bitter aftertaste is able to influence affective ratings and the neuronal processing of visual food cues. We presented 39 healthy normal-weight women (mean age: 22.5 years) with images depicting high-caloric meat dishes, high-caloric sweets, and low-caloric vegetables after they had either rinsed their mouth with wormwood tea (bitter group; n = 20) or water (control group; n = 19) for 30s. The bitter aftertaste of wormwood enhanced fronto-central early potentials (N100, N200) and reduced P300 amplitudes for all food types (meat, sweets, vegetables). Moreover, meat and sweets elicited higher fronto-central LPPs than vegetables in the water group. This differentiation was absent in the bitter group, which gave lower arousal ratings for the high-caloric food. We found that a minor intervention ('bitter rinse') was sufficient to induce changes in the neuronal processing of food images reflecting increased early attention (N100, N200) as well as reduced affective value (P300, LPP). Future studies should investigate whether this intervention is able to influence eating behavior.