Liquid Profiling for Cancer Patient Stratification in Precision Medicine-Current Status and Challenges for Successful Implementation in Standard Care.

Circulating tumor DNA (ctDNA), accurately described by the term liquid profiling (LP), enables real-time assessment of the tumor mutational profile as a minimally invasive test and has therefore rapidly gained traction, particular for the management of cancer patients. By LP, tumor-specific genetic alterations can be determined as part of companion diagnostics to guide selection of appropriate targeted therapeutics. Because LP facilitates longitudinal monitoring of cancer patients, it can be used to detect acquired resistant mechanisms or as a personalized biomarker for earlier detection of disease recurrence, among other applications. However, LP is not yet integrated into routine care to the extent that might be expected. This is due to the lack of harmonization and standardization of preanalytical and analytical workflows, the lack of proper quality controls, limited evidence of its clinical utility, heterogeneous study results, the uncertainty of clinicians regarding the value and appropriate indications for LP and its interpretation, and finally, the lack of reimbursement for most LP tests. In this review, the value proposition of LP for cancer patient management and treatment optimization, the current status of implementation in standard care, and the main challenges that need to be overcome are discussed in detail.

Liquid profiling of circulating tumor DNA in colorectal cancer: steps needed to achieve its full clinical value as standard care.

The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid profiling (LP), its acceptance by clinicians, and its integration into clinical workflows in real-world settings remain limited. Here, LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at < 0.1% in over 20% of cases, a high frequency of concomitant driver mutations (in up to 14% of cases), and ctDNA levels reflecting the clinical course of disease were revealed. However, certain limitations hampering successful translation of ctDNA into clinical practice were uncovered, including the lack of clinically relevant ctDNA thresholds, appropriate time points of LP requests, and integrative evaluation of ctDNA, imaging, and clinical findings. In conclusion, these results highlight the potential clinical value of LP for CRC patient management and demonstrate issues that need to be addressed for successful long-term implementation in clinical workflows.

Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human plasma by LC-MS.

OBJECTIVES: Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30-140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids.Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins. DESIGN AND METHODS: We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes.We compared the proteins detected by UC alone and UC coupled with SEC. RESULTS: In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone. CONCLUSIONS: We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.

Mass spectrometry-based method for quantification of nimodipine and glutamate in cerebrospinal fluid. Pilot study with patients after aneurysmal subarachnoid haemorrhage.

WHAT IS KNOWN AND OBJECTIVE: Delayed cerebral ischaemia is an important cause of morbidity and mortality after aneurysmal subarachnoid haemorrhage (aSAH). Nimodipine is the only drug approved by the FDA for improving outcome after aSAH. Clinically, however, there are no specific values of this drug in cerebrospinal fluid (CSF) during aSAH treatment that could be associated to outcome improvement. Furthermore, the neurotransmitter glutamate acts as a secondary marker for brain injury. The aim was to establish a method to measure nimodipine and glutamate concentrations simultaneously in CSF of patients after aSAH. METHODS: From June 2017 to June 2018, we prospectively collected clinical data of patients with aSAH admitted to our neurointensive care unit. All included patients received nimodipine orally (60 mg every 4 hours). Patients, who developed clinical vasospasm during their in-hospital stay, underwent intra-arterial application of nimodipine (IAN), followed by angiographic control. A method using high-performance liquid chromatography coupled with mass spectrometric analysis (LC-MS/MS) was established for quantification of both analytes in CSF. RESULTS AND DISCUSSION: In 15 (60%) of 25 patients, nimodipine and glutamate concentrations were measured. After IAN for treatment of vasospasms, CSF nimodipine concentrations were slightly higher than in patients who received nimodipine only orally (0.60 ± 0.27 ng/mL vs 0.48 ± 0.18 ng/mL). Patients developing vasospasm exhibited higher glutamate concentrations than patients without vasospasm (188.84 ng/mL vs136.07 ng/mL). WHAT IS NEW AND CONCLUSION: The developed method allowed the simultaneous quantification of nimodipine and glutamate in CSF. Furthermore, we demonstrated that IAN resulted in higher concentrations in CSF, when compared to oral application only.

Therapeutic drug monitoring (TDM) of 5-fluorouracil (5-FU): new preanalytic aspects.

Background 5-Fluorouracil (5-FU) is frequently used for the treatment of gastrointestinal tumors. The pharmacological effect of 5-FU is influenced by genetic polymorphisms as well as differently dosed regimens. Currently, 5-FU is generally administered as a continuous infusion via an implanted port system using a body surface area (BSA)-based dose calculation. In order to optimize treatment, the area under the curve (AUC) can be estimated to allow for individual dose adjustment. A 5-FU AUC range between 20 and 30 [mg×h×L] is recommended. The aim of the current study was to assess if blood for AUC analysis could also be drawn at the side where the port system had been placed. Methods We collected EDTA blood samples of patients receiving infusional 5-FU simultaneously from different sampling points (right/left cubital vein). 5-FU concentrations were measured in a steady-state equilibrium based on nanoparticle immunoassay (My5-FU; Saladax). Results A total of 39 patients took part in this study. About half of the patients did not reach the target 5-FU concentration window (37% were under- and 16% of the patients were overdosed). Calculated median AUC was 23.3 for the right arm (range 5.8-59.4) and a median of 23.4 for the left arm (range 5.3-61.0). AUC values showed no difference between right compared to left arms (p=0.99). Conclusions In all, these results confirm that a high percentage of patients are not treated with 5-FU doses reaching suggested AUC levels of 20-30. The location of venepuncture, however, had no impact on the results of plasma 5-FU concentration.

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions.

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein-protein interactions with other regulators such as phytochrome (FphA) or velvet-like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi-phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild-type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V-T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.

Light-dependent gene activation in Aspergillus nidulans is strictly dependent on phytochrome and involves the interplay of phytochrome and white collar-regulated histone H3 acetylation.

The ability for light sensing is found from bacteria to humans but relies only on a small number of evolutionarily conserved photoreceptors. A large number of fungi react to light, mostly to blue light. Aspergillus nidulans also responds to red light using a phytochrome light sensor, FphA, for the control of hundreds of light-regulated genes. Here, we show that photoinduction of one light-induced gene, ccgA, occurs mainly through red light. Induction strictly depends on phytochrome and its histidine-kinase activity. Full light activation also depends on the Velvet protein, VeA. This putative transcription factor binds to the ccgA promoter in an fphA-dependent manner but independent of light. In addition, the blue light receptor LreA binds to the ccgA promoter in the dark but is released after blue or red light illumination and together with FphA modulates gene expression through histone H3 modification. LreA interacts with the acetyltransferase GcnE and with the histone deacetylase HdaA. ccgA induction is correlated to an increase of the acetylation level of lysine 9 in histone H3. Our results suggest regulation of red light-induced genes at the transcriptional level involving transcription factor(s) and epigenetic control through modulation of the acetylation level of histone H3.

Fungi, hidden in soil or up in the air: light makes a difference.

Light is one of the most important environmental factors for orientation of almost all organisms on Earth. Whereas light sensing is of crucial importance in plants to optimize light-dependent energy conservation, in nonphotosynthetic organisms, the synchronization of biological clocks to the length of a day is an important function. Filamentous fungi may use the light signal as an indicator for the exposure of hyphae to air and adapt their physiology to this situation or induce morphogenetic pathways. Although a yes/no decision appears to be sufficient for the light-sensing function in fungi, most species apply a number of different, wavelength-specific receptors. The core of all receptor types is a chromophore, a low-molecular-weight organic molecule, such as flavin, retinal, or linear tetrapyrrols for blue-, green-, or red-light sensing, respectively. Whereas the blue-light response in fungi is one of the best-studied light responses, all other light-sensing mechanisms are less well studied or largely unknown. The discovery of phytochrome in bacteria and fungi in recent years not only advanced the scientific field significantly, but also had great impact on our view of the evolution of phytochrome-like photoreceptors.