A novel approach for BCR-ABL1 standardization to improve International Scale estimation.

INTRODUCTION: Standardization of BCR-ABL1 messenger RNA quantification by real-time PCR on the International Scale (IS) is critical for monitoring therapy response in chronic myelogenous leukaemia. Since 2006, BCR-ABL1 IS standardization is propagated along reference laboratories by calculating a laboratory-specific conversion factor (CF), co-ordinated in Europe through the European Treatment and Outcome Study project. Although this process has proven successful to some extent, it has not been achievable for all laboratories due to the complexity of the process and the stringent requirements in terms of numbers of samples to be exchanged. In addition, several BCR-ABL1 IS quantification methods and secondary reference materials became commercially available. However, it was observed that different IS methods generate consistently different results. METHODS: To overcome these difficulties, we have developed an alternative and simple approach of CF calculation, based on the retrospective analysis of existing external quality assessment (EQA) data. Our approach does not depend on the exchange of samples and is solely based on the mathematical CF calculation using EQA results. RESULTS AND CONCLUSION: We have demonstrated by thorough statistical validation that this approach performs well in converting BCR-ABL1 measurements to improve IS estimation. In expectation of a true golden standard method for BCR-ABL1 IS quantification, the proposed method is a valuable alternative.

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).

Reflections and proposals to assure quality in molecular diagnostics.

Molecular diagnostic testing has become an important tool in clinical laboratories. Accreditation according to the international quality standard ISO15189:2007 for medical laboratories is required for reimbursement of several molecular diagnostic tests in Belgium. Since the ISO15189:2007 standard applies to medical laboratories in general, the particular requirements for quality and competence are mentioned in general terms, not taking into account the specificities of molecular biology testing. Therefore, the working group "MolecularDiagnostics.be" described a consensus interpretation of chapter 5, Technical requirements, of the ISO standard for application in molecular diagnostic laboratories. The manuscript can be used as an instrument to prepare internal and external audits that meet the 15015189:2007 (chapter 5) criteria.

Thrombophilic factors do not predict outcomes in renal transplant recipients under prophylactic acetylsalicylic acid.

A cohort of recipients of renal transplant after 2000 (N=310) was prospectively screened on the day of transplantation and 1 month later for a panel of 11 thrombophilic factors to assess their effect on posttransplant outcomes. All patients received prophylactic acetylsalicylic acid, started before transplantation. The rate of thromboembolic events or acute rejection episodes during the first posttransplant year (primary composite endpoint) was 16.7% among patients free of thrombophilic factor (N=60) and 17.2% in those with >or=1 thrombophilic factor (N=250) (p>0.99). The incidence of the primary endpoint was similar among patients free of thrombophilic factors and those with >or=2 (N=135), or >or=3 (N=53) factors (16.3% and 15.1% respectively; p=1) and in patients who remained thrombophilic at 1 month (15.7%; p=0.84). None of the individual thrombophilic factor present at the day of transplantation was associated with the primary endpoint. The incidence of cardiovascular events at 1-year, serum creatinine at 1-year, 4-year actuarial graft and patient survival were not influenced by the presence of >or=1 thrombophilic factor at baseline (p=NS). In conclusion, the presence of thrombophilic factors does not influence thromboembolic events, acute rejection, graft or patient survival in patients transplanted after 2000 and receiving prophylactic acetylsalicylic acid.

Guidelines for an integrated diagnostic approach of chronic lymphoproliferative disorders in the routine laboratory of haematology in Belgium.

This paper summarizes the minimal workout of chronic lymphoproliferative disorders in a routine laboratory of haematology as recommended by a team of experienced laboratory supervisors in Belgium, taking into account the specific organisation of healthcare in Belgium, the innovations in the field of molecular analyses and related reimbursement. The starting point was essentially based upon clinical and/or haematological indications and it is emphasized that conclusions should be drawn in close dialogue with the clinician and experts in cytogenetics and histopathology. Reports made in the laboratory should be based upon an integration of cytomorphological, immunophenotypical and molecular data. These guidelines are not intended to be used as universal 'diagnostic pathways', but should be useful in developing local diagnostic pathways. It is well understood that this consensus, being valid anno 2009, may rapidly change with new technologies being introduced and new targets discovered.

Doubling time of soluble CD23: a powerful prognostic factor for newly diagnosed and untreated stage A chronic lymphocytic leukemia patients.

Soluble CD23 (sCD23) levels correlate with the stage, prognosis and overall survival (OS) of patients with chronic lymphocytic leukemia (CLL). Therefore, we prospectively evaluated sCD23 doubling time (sCD23DT) as a prognostic factor for time to treatment (TTT) and OS in 56 newly diagnosed and untreated CLL patients at Binet stage A, and compared it to the most commonly used biological prognostic factors: lymphocyte doubling time, immunoglobulin variable heavy chain (IgVH) mutational status and zeta-associated protein-70 (ZAP-70), CD38, and lipoprotein lipase (LPL) expression. In patients with sCD23DT <1 year, the median TTT and OS were 20 and 83 months compared to 141 and 177 months in patients with sCD23DT >1 year (P<0.0001). Among patients with poor prognostic factors (ZAP-70+, LPL+ and CD38+), an sCD23DT <1 year identified a subpopulation with a shorter TTT. Patients with unmutated IgVH and an sCD23DT <1 year had a median TTT and OS of 14 and 83 months, respectively, whereas these values were 70 and >177 months when sCD23DT was >1 year (P<0.0001 and P=0.0219, respectively). Finally, in a Cox multivariate analysis, sCD23DT was the sole independent prognostic factor for TTT (P=0.0027). Furthermore, sCD23DT refines the prognosis given by other classical prognostic factors. These observations support the introduction of sCD23 evaluation into the routine assessment of CLL patients.

Biclonal low grade B-cell lymphoma confirmed by both flow cytometry and karyotypic analysis, in spite of a normal kappa/lambda Ig light chain ratio.

Composite low grade lymphoma with two subpopulations in a same site is uncommon. We herewith report the case of an 80-year-old woman who presented with isolated bilateral dacryoadenomegaly. Pathological examination of an incisional biopsy of her right lacrimal gland was consistent with a marginal zone lymphoma. Flow cytometry immunophenotyping showed two distinct clonal B-cell populations expressing sIg D lambda or sIg M kappa restriction in the lacrimal gland, blood, and bone marrow. Both B-cells populations were sorted from peripheral blood for molecular biology investigations and comparison with molecular data performed on tumor and bone marrow cells. IgH PCR performed on purified blood populations disclosed two monoclonal peaks: 98 bp-sized peak in the sIg M kappa and a 107 bp in the sIg D lambda clones, respectively. The lacrimal gland tumor expressed mainly sIg M kappa population, and showed a major 98 bp-sized peak coexisting with a very minor 107 bp peak. Cytogenetic studies showed a 46, XX,del (7) (q22q32) karyotype. Bone marrow examination at diagnosis revealed the same B-cell clones distribution than the one observed in blood with a dominant sIg D lambda population, a Genescan profile showing a major peak of 107 bp and a minor peak of 98 bp. Chromosomal analysis disclosed a 46,XX,del (10) (?p14) karyotype without detectable 7q deletion. To our knowledge, this observation represents the first reported case of biclonal low grade lymphoma hidden behind a normal classical kappa/lambda Ig light chain ratio in blood, but clearly demonstrated by the combination of three ancillary techniques (flow cytometry both analytical and cell sorting, molecular biology, and cytogenetics) and analysis of different tissues (i.e., in this case, lacrimal gland biopsy, blood, and bone marrow).

Deficient host-bacteria interactions in inflammatory bowel disease? The toll-like receptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease and ulcerative colitis.

BACKGROUND AND AIMS: Elicitation of an innate immune response to bacterial products is mediated through pattern recognition receptors (PRRs) such as the toll-like receptors (TLRs) and the NODs. The recently characterised Asp299Gly polymorphism in the lipopolysaccharide (LPS) receptor TLR4 is associated with impaired LPS signalling and increased susceptibility to Gram negative infections. We sought to determine whether this polymorphism was associated with Crohn's disease (CD) and/or ulcerative colitis (UC). METHODS: Allele frequencies of the TLR4 Asp299Gly polymorphism and the three NOD2/CARD15 polymorphisms (Arg702Trp, Gly908Arg, and Leu1007fsinsC) were assessed in two independent cohorts of CD patients (cohort 1, n = 334; cohort 2, n = 114), in 163 UC patients, and in 140 controls. A transmission disequilibrium test (TDT) was then performed on 318 inflammatory bowel disease (IBD) trios. RESULTS: The allele frequency of the TLR4 Asp299Gly polymorphism was significantly higher in CD (cohort 1: 11% v 5%, odds ratio (OR) 2.31 (95% confidence interval (CI) 1.28-4.17), p = 0.004; and cohort 2: 12% v 5%, OR 2.45 (95% CI 1.24-4.81), p = 0.007) and UC patients (10% v 5%, OR 2.05 (95% CI 1.07-3.93), p = 0.027) compared with the control population. A TDT on 318 IBD trios demonstrated preferential transmission of the TLR4 Asp299Gly polymorphism from heterozygous parents to affected children (T/U: 68/34, p = 0.01). Carrying polymorphisms in both TLR4 and NOD2 was associated with a genotype relative risk (RR) of 4.7 compared with a RR of 2.6 and 2.5 for TLR4 and NOD2 variants separately. CONCLUSION: We have reported on a novel association of the TLR4 Asp299Gly polymorphism with both CD and UC. This finding further supports the genetic influence of PRRs in triggering IBD.

[Medical genetics service]. / Le Service de Génétique Médicale.

Created in 1987, the department of medical genetics finds its origins in molecular endocrinology research which had developed from the seventies at the Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) of the Faculty of Medicine. After its fusion with the Center of Human Genetics of the ULB, in 1992, the department is composed of three units: the lab of molecular genetics and oncology, the lab of cytogenetics and a clinical genetics unit. One thousand consultations of genetic counseling and more than 15,000 molecular or cytogenetic diagnostic procedures are performed annually. The development of the clinical activities was paralleled by a very active research activity, resulting in a series of "firsts". Amongst the main results are: the identification of the first mutations responsible for congenital hypothyroidism; the molecular cloning of the TSH receptor and of a series of "orphan" G protein-coupled receptors; the identification of a novel neuropeptide, nociceptin, by the first example of "reverse pharmacology"; the identification of olfactory receptors on the sperm of mammals, including man; the identification in molecular terms of the mechanisms responsible for acquired and hereditary hyperthyroidisms; the identification of the chemokine receptor CCR5 as the major coreceptor of HIV-1, and of the prevalent mutation of CCR5 conferring resistance to HIV to about 1% of the European population.

Fusion of a novel gene, RCC17, to the TFE3 gene in t(X;17)(p11.2;q25.3)-bearing papillary renal cell carcinomas.

A subset of childhood and young adult renal cell carcinomas displays a recurrent translocation t(X;17)(p11;q25) as the sole cytogenetic abnormality. In two young girls, we demonstrate that this translocation results in the fusion of a novel gene, designated RCC17, at chromosome 17q25, to the transcription factor TFE3 located on the Xp11 chromosomal region. In both cases, the t(X;17) fuses the NH(2)-terminal region of RCC17 to the COOH-terminal part of TFE3 including the basic helix-loop-helix DNA-binding domain and the leucine zipper dimerization domain. The reciprocal fusion transcript TFE3/RCC17 is also expressed. RCC17 encodes a putative protein of 553 amino acids. It is ubiquitously expressed in normal adult tissues. No significant similarity was found with other fusion partners of TFE3 or with any relevant functional protein domains, precluding informed speculation about the normal function of this gene.

Use of two-hybrid methodology for identifying proteins of interest in endocrinology.

During the last 10 years, much progress has been made in understanding signal transduction. However, the function of many newly identified proteins remains unknown. The protein/protein interactions have emerged as a major biochemical mechanism of signal transduction. They are of major interest to elucidate the role of a protein in one or another cellular process. The two-hybrid system is especially well designed for such investigation. Here we show that the contribution of this technique already is and will be essential in dissecting the molecular mechanism of transduction pathways in many cell types.

Cloning and sequence analysis of human calcyphosine complementary DNA.

Calcyphosine, initially identified as thyroid protein p24, is a calcium-binding protein containing four EF-hand domains. It was first cloned and characterized in the dog and corresponds to R2D5 antigen in rabbit. Using the canine calcyphosine cDNA sequence as a probe, we have isolated its human counterpart from a thyroid cDNA library. The two sequences display a high degree of conservation, both at nucleotide and deduced amino acid levels. Sequence comparison with other proteins showed that the closest homologue of calcyphosine is the crustacean CCBP-23 protein. Northern blot analysis revealed that calcyphosine messenger RNA is much less abundant in human than in canine thyrocytes. Western blot experiments indicated that the amount of protein is also dramatically reduced in man compared to dog.

Production of dog calcyphosine in bacteria and lack of phosphorylation by the catalytic subunit of protein kinase A in vitro.

Calcyphosine is a calcium-binding protein containing four EF-hand domains that is found in several epithelia and in some cells of the central nervous system. In thyroid follicular cells, calcyphosine is synthesized and phosphorylated in response to stimulation by thyrotropin and cAMP agonists. The cDNA coding for dog calcyphosine has been expressed in bacteria under the control of the T7 promoter. Recombinant calcyphosine was purified from crude bacterial lysates by a combination of anion-exchange and hydrophobic interaction chromatography. Phosphorylation assays using the purified catalytic subunit of protein kinase A and the recombinant or the native calcyphosine revealed that, contrary to a previous report, calcyphosine is not significantly phosphorylated by this enzyme in vitro.