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
BACKGROUND: Cluster headache is a severe headache disorder with unknown aetiology. The pathophysiology and symptoms present certain common features with migraine. Specifically, activation of the trigeminal vascular system seems to be involved in both disorders, which is hypothesized to result in neurogenic inflammation and vasodilation of the cerebral vessels. In addition, genetic factors have been implicated in both migraine and cluster headache. OBJECTIVE: In order to determine whether or not migraine and cluster headache share genetic risk factors, we screened two genetic variants known to increase the risk of migraine in Sweden in a Swedish cluster headache case-control study population. METHODS: In all, 541 patients and 581 control subjects were genotyped for rs1835740 in close proximity to MTDH (metadherin) and rs2651899 in the PRDM16 (PR/SET domain 16) gene, using TaqMan® real-time PCR and pyrosequencing. In addition, we analyzed MTDH gene expression in a subset of the material, using reverse transcription real-time PCR to determine relative mRNA levels in primary fibroblast cell lines from patients and controls. RESULTS: We found a trend for association between rs1835740, which is reported to affect MTDH mRNA levels, and cluster headache in our Swedish case-control material (p = 0.043, Χ2 = 4.102). This association was stronger in a subgroup of patients suffering from both cluster headache and migraine (p = 0.031, Χ2 = 6.964). We could further confirm that rs1835740 has an effect on the transcriptional activity of MTDH. In this Swedish cluster headache cohort we did not find an association with the rs2651899 variant. CONCLUSIONS: We conclude that rs1835740 is a potential risk factor for cluster headache in Sweden. Our data indicates that rs1835740 and MTDH might be involved in neurovascular headaches in general whilst rs2651899 is specifically related to migraine.
Subject(s)
Cell Adhesion Molecules/genetics , Cluster Headache/epidemiology , Cluster Headache/genetics , Genetic Predisposition to Disease/epidemiology , Genetic Predisposition to Disease/genetics , Polymorphism, Single Nucleotide/genetics , Adult , Case-Control Studies , Cluster Headache/diagnosis , Cohort Studies , Female , Genetic Variation/genetics , Genome-Wide Association Study/methods , Humans , Male , Membrane Proteins , Middle Aged , Migraine Disorders/diagnosis , Migraine Disorders/epidemiology , Migraine Disorders/genetics , Population Surveillance/methods , RNA-Binding Proteins , Risk Factors , Sweden/epidemiologyABSTRACT
Background Cluster headache is characterized by recurrent unilateral headache attacks of severe intensity. One of the main features in a majority of patients is a striking rhythmicity of attacks. The CLOCK ( Circadian Locomotor Output Cycles Kaput) gene encodes a transcription factor that serves as a basic driving force for circadian rhythm in humans and is therefore particularly interesting as a candidate gene for cluster headache. Methods We performed an association study on a large Swedish cluster headache case-control sample (449 patients and 677 controls) screening for three single nucleotide polymorphisms (SNPs) in the CLOCK gene implicated in diurnal preference (rs1801260) or sleep duration (rs11932595 and rs12649507), respectively. We further wanted to investigate the effect of identified associated SNPs on CLOCK gene expression. Results We found a significant association with rs12649507 and cluster headache ( p = 0.0069) and this data was strengthened when stratifying for reported diurnal rhythmicity of attacks ( p = 0.0009). We investigated the effect of rs12649507 on CLOCK gene expression in human primary fibroblast cultures and identified a significant increase in CLOCK mRNA expression ( p = 0.0232). Conclusions Our results strengthen the hypothesis of the involvement of circadian rhythm in cluster headache.
