Analysis of measurable residual disease by IG/TR gene rearrangements: quality assurance and updated EuroMRD guidelines.

Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).

Patient specific real-time PCR in precision medicine - Validation of IG/TR based MRD assessment in lymphoid leukemia.

Detection of patient- and tumor-specific clonally rearranged immune receptor genes using real-time quantitative (RQ)-PCR is an accepted method in the field of precision medicine for hematologic malignancies. As individual primers are needed for each patient and leukemic clone, establishing performance specifications for the method faces unique challenges. Results for series of diagnostic assays for CLL and ALL patients demonstrate that the analytic performance of the method is not dependent on patients' disease characteristics. The calibration range is linear between 10-1 and 10-5 for 90% of all assays. The detection limit of the current standardized approach is between 1.8 and 4.8 cells among 100,000 leukocytes. RQ-PCR has about 90% overall agreement to flow cytometry and next generation sequencing as orthogonal methods. Accuracy and precision across different labs, and above and below the clinically applied cutoffs for minimal/measurable residual disease (MRD) demonstrate the robustness of the technique. The here reported comprehensive, IVD-guided analytical validation provides evidence that the personalized diagnostic methodology generates robust, reproducible and specific MRD data when standardized protocols for data generation and evaluation are used. Our approach may also serve as a guiding example of how to accomplish analytical validation of personalized in-house diagnostics under the European IVD Regulation.

The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach.

Dehydration leads to different physiological and biochemical responses in plants. We analysed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation-tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant) and two desiccation-sensitive species, Lindernia subracemosa and Arabidopsis thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolysed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x-DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x-DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation-sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer- to non-bilayer-forming lipids, thus contributing to protein and membrane stabilization.

Quantification of diacylglycerol by mass spectrometry.

Diacylglycerol (DAG) is an important intermediate of lipid metabolism and a component of phospholipase C signal transduction. Quantification of DAG in plant membranes represents a challenging task because of its low abundance. DAG can be measured by direct infusion mass spectrometry (MS) on a quadrupole time-of-flight mass spectrometer after purification from the crude plant lipid extract via solid-phase extraction on silica columns. Different internal standards are employed to compensate for the dependence of the MS and MS/MS signals on the chain length and the presence of double bonds in the acyl moieties. Thus, using a combination of single MS and MS/MS experiments, quantitative results for the different molecular species of DAGs from Arabidopsis can be obtained.

Suberin goes genomics: use of a short living plant to investigate a long lasting polymer.

Suberin is a highly persistent cell wall polymer, predominantly composed of long-chain hydroxylated fatty acids. Apoplastic suberin depositions occur in internal and peripheral dermal tissues where they generate lipophilic barriers preventing uncontrolled flow of water, gases, and ions. In addition, suberization provides resistance to environmental stress conditions. Despite this physiological importance the knowledge about suberin formation has increased slowly for decades. Lately, the chemical characterization of suberin in Arabidopsis enabled the proposal of genes required for suberin biosynthesis such as ß-ketoacyl-CoA synthases (KCS) for fatty acid elongation and cytochrome P450 oxygenases (CYP) for fatty acid hydroxylation. Advantaged by the Arabidopsis molecular genetic resources the in silico expression pattern of candidate genes, concerted with the tissue-specific distribution of suberin in Arabidopsis, led to the identification of suberin involved genes including KCS2, CYP86A1, and CYP86B1. The isolation of mutants with a modified suberin composition facilitated physiological studies revealing that the strong reduction in suberin in cyp86a1 mutants results in increased root water and solute permeabilities. The enhanced suberin 1 mutant, characterized by twofold increased root suberin content, has increased water-use efficiency and is affected in mineral ion uptake and transport. In this review the most recent findings on the biosynthesis and physiological importance of suberin in Arabidopsis are summarized and discussed.

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry.

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

A processive glycosyltransferase involved in glycolipid synthesis during phosphate deprivation in Mesorhizobium loti.

Natural habitats are often characterized by a low availability of phosphate. In plants and many bacteria, phosphate deficiency causes different physiological responses, including the replacement of phosphoglycerolipids in the membranes with nonphosphorous lipids. We describe here a processive glycosyltransferase (Pgt) in Mesorhizobium loti (Rhizobiales) involved in the synthesis of di- and triglycosyldiacylglycerols (DGlycD and TGlycD) during phosphate deprivation. Cells of the corresponding Δpgt deletion mutant are deficient in DGlycD and TGlycD. Additional Pgt-independent lipids accumulate in Mesorhizobium after phosphate starvation, including diacylglyceryl trimethylhomoserine (DGTS) and ornithine lipid (OL). The accumulation of the nonphosphorous lipids during phosphate deprivation leads to the reduction of phosphoglycerolipids from 90 to 50%. Nodulation experiments of Mesorhizobium wild type and the Δpgt mutant with its host plant, Lotus japonicus, revealed that DGlycD and TGlycD are not essential for nodulation under phosphate-replete or -deficient conditions. Lipid measurements showed that the Pgt-independent lipids including OL and DGTS accumulate to higher proportions in the Δpgt mutant and therefore might functionally replace DGlycD and TGlycD during phosphate deprivation.