ABSTRACT
BACKGROUND: Liquid biopsies are emerging as valuable clinical biomarkers for cancer monitoring. Although International Organization for Standards (ISO) and Technical Specifications from the European Committee for Standardization (CEN/TS) standardized workflows exist, their implementation in clinical practice is underdeveloped. We aimed to assess the applicability of ISO and CEN/TS standards in a real-world clinical setting, with a particular focus on evaluating the impact of preanalytical parameters and hemolysis on liquid biopsy analysis. METHODS: We evaluated 659 peripheral blood samples from advanced prostate cancer patients against ISO and CEN/TS standards and documented all essential criteria, including tube draw order, filling level, temperature, and time tracking from blood draw to storage. We assessed hemolysis and its effect on circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) analysis. RESULTS: Our results demonstrated a high compliance rate, with 96.2% (634/659) of samples meeting essential ISO and CEN/TS criteria. We did not observe a significant impact on ctDNA or CTC detection rates between hemolytic and nonhemolytic samples. Hemolysis was identified in 12.9% (40/311) of plasma samples from our advanced prostate cancer cohort, and within the draw order of 5 blood collection tubes, hemolysis did not significantly increase from tube 1 to 5. In total, 83.8% (552/659) of blood collection tubes had high fill levels above 80% of nominal filling level. CONCLUSIONS: Our study demonstrates the feasibility and benefits of adhering to ISO and CEN/TS standards in a clinical liquid biopsy study. The standards revealed that hemolysis occurred frequently but did not impair downstream ctDNA and CTC analysis in our cohort of advanced prostate cancer patients.
ABSTRACT
Attenuation of cell-free DNA clearance in vivo is an alternative strategy to maximize recovery.
Subject(s)
Biomarkers, Tumor , Cell-Free Nucleic Acids , Biomarkers, Tumor/blood , Biomarkers, Tumor/chemistry , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/chemistry , Liquid Biopsy , Humans , Animals , Mice , Sensitivity and SpecificityABSTRACT
Alterations of the androgen receptor (AR) are associated with resistance to AR-directed therapy in prostate cancer. Thus, it is crucial to develop robust detection methods for AR alterations as predictive biomarkers to enable applicability in clinical practice. We designed and validated five multiplex droplet digital PCR assays for reliable detection of 12 AR targets including AR amplification, AR splice variant 7, and 10 AR hotspot mutations, as well as AR and KLK3 gene expression from plasma-derived cell-free DNA and cell-free RNA. The assays demonstrated excellent analytical sensitivity and specificity ranging from 95% to 100% (95% CI, 75% to 100%). Intrarun and interrun variation analyses revealed a high level of repeatability and reproducibility. The developed assays were applied further in peripheral blood samples from 77 patients with advanced prostate cancer to assess their feasibility in a real-world scenario. Optimizing the reverse transcription of RNA increased the yield of plasma-derived cell-free RNA by 30-fold. Among 23 patients with castration-resistant prostate cancer, 6 patients (26.1%) had one or a combination of several AR alterations, whereas only 2 of 54 patients (3.7%) in the hormone-sensitive stage showed AR alterations. These findings were consistent with other studies and suggest that implementation of comprehensive AR status detection in clinical practice is feasible and can support the treatment decision-making process.
Subject(s)
Receptors, Androgen , Humans , Receptors, Androgen/genetics , Male , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , Aged , Prostatic Neoplasms/genetics , Prostatic Neoplasms/diagnosis , Reproducibility of Results , Mutation , Sensitivity and Specificity , Middle Aged , Cell-Free Nucleic Acids/genetics , Cell-Free Nucleic Acids/blood , Kallikreins/blood , Kallikreins/genetics , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/diagnosis , Prostatic Neoplasms, Castration-Resistant/blood , Multiplex Polymerase Chain Reaction/methodsABSTRACT
The advancements in the detection and characterization of circulating tumor DNA (ctDNA) have revolutionized precision medicine and are likely to transform standard clinical practice. The non-invasive nature of this approach allows for molecular profiling of the entire tumor entity, while also enabling real-time monitoring of the effectiveness of cancer therapies as well as the identification of resistance mechanisms to guide targeted therapy. Although the field of ctDNA studies offers a wide range of applications, including in early disease, in this review we mainly focus on the role of ctDNA in the dynamic molecular characterization of unresectable locally advanced and metastatic BC (mBC). Here, we provide clinical practice guidance for the rapidly evolving field of molecular profiling of mBC, outlining the current landscape of liquid biopsy applications and how to choose the right ctDNA assay. Additionally, we underline the importance of exploring the clinical relevance of novel molecular alterations that potentially represent therapeutic targets in mBC, along with mutations where targeted therapy is already approved. Finally, we present a potential roadmap for integrating ctDNA analysis into clinical practice.
ABSTRACT
In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans.
Subject(s)
Insulin Resistance , Intermittent Fasting , Tumor Suppressor Protein p53 , Animals , Humans , Mice , Adipocytes/metabolism , Adipose Tissue/metabolism , Inflammation/metabolism , Insulin Resistance/genetics , Obesity/genetics , Obesity/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Weight LossABSTRACT
Liquid biopsy for minimally invasive diagnosis and monitoring of cancer patients is progressing toward routine clinical practice. With the implementation of highly sensitive next-generation sequencing (NGS) based assays for the analysis of cfDNA, however, consideration of the utility of liquid biopsy for clinical genetic testing is critical. While the focus of liquid biopsy for cancer diagnosis is the detection of circulating tumor DNA (ctDNA) as a fraction of total cell-free DNA (cfDNA), cfDNA analysis reveals both somatic mosaic tumor and germline variants and clonal hematopoiesis. Here we outline advantages and limitations of mosaic and germline variant detection as well as the impact of clonal hematopoiesis on liquid biopsy in cancer diagnosis. We also evaluate the potential of cfDNA analysis for the molecular diagnosis of monogenic mosaic disorders.