Molecular data show conserved DNA locations distinguishing lung cancer subtypes and regulation of immune genes

Introduction: Non-small-cell lung cancer exhibits a range of transcriptional and epigenetic patterns that not only define distinct phenotypes, but may also govern immune related genes, which have a major impact on survival. Methods: We used open-source RNA expression and DNA methylation data of the Cancer Genome Atlas with matched non-cancerous tissue to evaluate whether these pretreatment molecular patterns also influenced genes related to the immune system and overall survival. Results: The distinction between lung adenocarcinoma and squamous c

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies

Strain partitioning in a large intracontinental strike-slip system accommodating backarc-convex orocline formation: The Circum-Moesian Fault System of the Carpatho-Balkanides

The evolution of oroclines is often driven by the interplay of subduction and indentation associated with complex patterns of deformation transfer from shortening to strike-slip and extension. We study the kinematics and mechanics of indentation in an orocline with a backarc-convex geometry, the European Carpatho-Balkanides Mountains. Within this orocline, the kinematic evolution of the Serbian Carpathians segment is less understood. The results demonstrate that the overall deformation was accommodated by the Circum-Moesian Fault System surrounding the Moesian indenter, where strain was partitioned in a complex network of coeval strike-slip, thrust and normal faults. This system represents one of the largest European intracontinental strike-slip deformation zones, with a northward-increasing accumulated 140 km dextral offset along previously known and newly found faults. These strike-slip faults transfer a significant part of their offset eastwards to thrusting in the Balkanides and westwards to orogen-parallel extension and the formation of intramontane basins. The correlation with paleogeographic and geodynamic reconstructions demonstrates that the overall formation of the fault system is driven by subduction of the Carpathian embayment, resulting in laterally variable amounts of translation and rotation associated with indentation of the Moesian Platform. The onset of Carpathian slab retreat and backarc extension at 20 Ma has dramatically increased the rates of dextral deformation from ~3.5 mm/yr to ~2 cm/yr, facilitated by the pull exerted by the retreating slab. Our study demonstrates that indentation requires a strain partitioning analysis that is adapted to the specificity of deformation mechanics and is, therefore, able to quantify the observed kinematic patterns

Strain partitioning in a large intracontinental strike-slip system accommodating backarc-convex orocline formation: The Circum-Moesian Fault System of the Carpatho-Balkanides

The evolution of oroclines is often driven by the interplay of subduction and indentation associated with complex patterns of deformation transfer from shortening to strike-slip and extension. We study the kinematics and mechanics of indentation in an orocline with a backarc-convex geometry, the European Carpatho-Balkanides Mountains. Within this orocline, the kinematic evolution of the Serbian Carpathians segment is less understood. The results demonstrate that the overall deformation was accommodated by the Circum-Moesian Fault System surrounding the Moesian indenter, where strain was partitioned in a complex network of coeval strike-slip, thrust and normal faults. This system represents one of the largest European intracontinental strike-slip deformation zones, with a northward-increasing accumulated 140 km dextral offset along previously known and newly found faults. These strike-slip faults transfer a significant part of their offset eastwards to thrusting in the Balkanides and westwards to orogen-parallel extension and the formation of intramontane basins. The correlation with paleogeographic and geodynamic reconstructions demonstrates that the overall formation of the fault system is driven by subduction of the Carpathian embayment, resulting in laterally variable amounts of translation and rotation associated with indentation of the Moesian Platform. The onset of Carpathian slab retreat and backarc extension at 20 Ma has dramatically increased the rates of dextral deformation from ~3.5 mm/yr to ~2 cm/yr, facilitated by the pull exerted by the retreating slab. Our study demonstrates that indentation requires a strain partitioning analysis that is adapted to the specificity of deformation mechanics and is, therefore, able to quantify the observed kinematic patterns

Mephenytoin as a probe for CYP2C19 phenotyping: effect of sample storage, intra-individual reproducibility and occurrence of adverse events

Aims To further evaluate mephenytoin as a probe for CYP2C19 phenotyping. Methods Healthy subjects (n=2638) were phenotyped using the urinary (S)-mephenytoin to (R)-mephenytoin ratio. This method was evaluated for (a) the stability of the S/R-ratio following sample storage, (b) the intraindividual reproducibility of the ratio, and (c) the occurrence of adverse events. Results After prolonged storage, the S/R-ratio of samples from extensive metabolisers (EM) increased up to 85%. In 1.5% of the cases (1 out 66), this led to incorrect classification of phenotype. In EMs, but not in poor metabolisers (PMs), the S/R-ratio increased after acid treatment. The intraindividual reproducibility of the mephenytoin phenotyping procedure was 28%. No major side-effects were observed and there was no relationship between the incidence of side-effects and the phenotype of the subject. Conclusions After prolonged storage the S/R-ratio significantly increased in EMs and, although low, the risk of incorrect classification should not be ignored. Our data support the use of mephenytoin as a safe drug for CYP2C19 phenotyping

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies

External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium

BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands).METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance.RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of &gt;0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately.CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.</p