Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Association of CT60 cytotoxic T lymphocyte antigen-4 gene polymorphism with thyroid autoantibody production in patients with Hashimoto's and postpartum thyroiditis.

Strong genetic contribution has been demonstrated to influence the development of autoimmune thyroid disease (AITD) as well as thyroid autoantibody production. In order to assess the relation between CT60 cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism and thyroid autoantibody production, we investigated 180 consecutive newly diagnosed patients with two forms of AITD, 105 with Hashimoto's thyroiditis (HT) and 75 with postpartum thyroiditis (PPT). We evaluated thyroid function, measured antibodies against thyroid peroxidase (TPO) and thyroglobulin (Tg), and determined CT60 CTLA-4 gene polymorphism. In HT, TPO antibody median value was significantly lower in the AA compared to the AG and GG genotypes (65, 122 and 319 U/ml, P<0.005), while the Tg antibody median value was lower in the AA compared to the AG genotype (91 and 189 U/ml, P<0.02). In PPT, the frequency of thyroid autoantibody-positive patients was higher among G-allele-carrying genotypes (P<0.04). Similar to HT, the TPO antibody median value was lower in the AA compared to the AG and GG genotypes (12, 130 and 423 U/ml, P<0.006). Hypothyroid PPT patients were more often thyroid autoantibody-positive (P<0.005) and the TPO antibody median value was higher compared to hyperthyroid PPT patients (500 and 32 U/ml, P<0.0001). The frequency of the G-allele was significantly higher among hypothyroid patients (P<0.05). Our data suggest that in both HT and PPT, the CT60 CTLA-4 gene polymorphism contributes importantly to thyroid autoantibody production. In PPT, the genotype also seems to influence thyroid function, as patients with the polymorphous allele are more prone to develop hypothyroid form of PPT.

Biotransformation of steroids by the fission yeast Schizosaccharomyces pombe.

The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- and regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal products. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone was reduced at the delta 4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and 4-androstene-3,17-dione were interconverted and 5 alpha-pregnane-3,20-dione and 5 beta-pregnane-3,20-dione were reduced to 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombe more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces cerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity.

Induction of steroidal hydroxylase activity by plant defence compounds in the filamentous fungus Cochliobolus lunatus

We investigated the hypothesis that the endogenous role of the commercially important inducible steroid hydroxylase cytochrome P450s of fungi was in defense against plant toxophores/secondary metabolites. Two plant defense compounds, the aglycones tomatidine and solanidine, the steroidal glycoalkaloid alpha-tomatine and the triterpene saponin beta-escin were tested as inducers of 11beta/14alpha-steroid hydroxylase in the filamentous fungus Cochliobolus lunatus. The extracts of saponins from the roots of Primula veris and green oat leaves were also tested as inducers of 11beta/14alpha-hydroxylation activity in progesterone biotransformation with the same fungus. Induction of steroid hydroxylase and inhibition of activity in some cases support our hypothesis that their endogenous function is in biochemical defence against secondary metabolites. 4-Pregnene-3,11,20-trione was added as a substrate for biotransformation with C. lunatus. We isolated from culture broth 14alpha-hydroxy-4-pregnene-3,11,20-trione, and the hitherto unreported compounds, 7alpha,14alpha-dihydroxy-4-pregnene-3,11,20-trione and 7alpha-hydroxy-pregna-4,8(14)-diene-3,11,20-trione.

11Beta-hydroxysteroid dehydrogenase activity in progesterone biotransformation by the filamentous fungus Cochliobolus lunatus.

Progesterone biotransformation was examined in relation to hydroxylating and dehydrogenating enzymes of Cochliobolus lunatus. 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) was located in cytosolic fraction and was NADP-dependent, inducible by progesterone and apparently uni-directional. Several inhibitors of 11beta-hydroxysteroid dehydrogenase were tested; furosemide, glycyrrhizic-acid and carbenoxolone did not influence the dehydrogenation of 11beta-hydroxy-4-pregnene-3,20-dione to 4-pregnene-3,11,20-trione, although grapefruit juice significantly reduced the rate of progesterone hydroxylation.