Serial quantitation of BCR-ABL IntroductionReverse-transcription real-time quantitative polymerase chain reaction (RQ-PCR) is used routinely to quantify levels of BCR-ABL mRNA in peripheral blood and bone marrow samples from chronic myelogenous leukemia (CML) patients undergoing therapy. The technique can accurately determine response to treatment and is particularly valuable for patients who have achieved a complete cytogenetic response. The National Comprehensive Cancer Network (NCCN) 1 and the European LeukemiaNet (ELN) 2 recommend similar monitoring schedules for patients treated with imatinib and the ELN defines an optimal response as the attainment of a major molecular response (MMR) after 18 months of therapy. Monitoring of BCR-ABL mRNA levels is also useful for gauging therapeutic response for patients with Philadelphia chromosomepositive acute lymphoblastic leukemia (Ph ϩ ALL). The CML meeting at the National Institutes of Health in Bethesda in October 2005 made several recommendations for the harmonization of minimal residual disease (MRD) assessment and proposed an international scale (IS) for BCR-ABL RQ-PCR measurements. 8 Importantly, the IS is essentially identical to that used in the International Randomized Study of Interferon and STI571 (IRIS) study, 9 with the IRIS standardized baseline defined as 100% BCR-ABL IS and MMR (3-log reduction relative to the standardized baseline) defined as 0.1% BCR-ABL IS . The original standards used for the IRIS trial are no longer available, however traceability to the IRIS scale is provided by the extensive quality control data generated by the Adelaide laboratory over a period of several years. 10,11 To enable testing centers to gain access to the IS, the Adelaide laboratory initiated a process to develop and validate laboratoryspecific conversion factors (CFs) that can be used to convert local values to IS values. 11 The strength of this approach is that testing centers can continue to use their existing assay conditions and continue to express results according to local preferences in addition to expressing results on the IS. The concept of the IS is analogous to established procedures for other quantitative assays, for example the International Normalized Ratio (INR) for prothrombin time. 12 Many laboratories with validated CFs have established themselves as national or regional reference laboratories and are in the process of propagating CFs to local centers. 13 While this process has generally worked well, it is apparent that the establishment of CFs is time-consuming, complex, expensive, and open to only a limited number of laboratories at any given time. Furthermore, it is unclear how frequently any individual CF will need to be revalidated. We sought therefore to develop an alternative means for testing laboratories to access the IS by developing calibrated, accredited primary reference reagents for BCR-ABL RQ-PCR analysis. StrategyIdeally, the formulation for primary reference reagents should be as close as possible to the usual analyte, should cove...

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 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 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).

AimsMyeloproliferative neoplasms (MPN) are a heterogeneous group of clonal proliferative bone marrow diseases characterised by extensive megakaryocytic hyperplasia and morphological atypia. Despite knowledge of genomic defects, the pathobiological processes driving these megakaryocytic abnormalities in MPN remain poorly explained. We have explored the proliferative, apoptotic and epigenetic profiles of megakaryocytes in human MPN.MethodsImmunohistochemical staining was performed on bone marrow trephine biopsies of 81 MPN (with and withoutJAK2V617FandCALRmutations) and 15 normal controls to assess the megakaryocytic expression of biomarkers associated with proliferation (Ki67), apoptosis (Bcl-XL, BNIP-3) and epigenetic regulation (EZH2, SUZ12).ResultsMyeloproliferative megakaryocytes showed significantly greater expression of proliferative Ki67 and anti-apoptotic Bcl-XL, reduced pro-apoptotic BNIP-3 and increased SUZ12 compared with controls. In essential thrombocythaemia, large-giant megakaryocytes with hyperlobated nuclei showed a trend towards a proliferative signature. In contrast, myelofibrotic megakaryocytes with condensed nuclear chromatin, and cases withCALRmutations, had significant reductions in pro-apoptotic BNIP-3.ConclusionsUncontrolled megakaryocytic expansion in MPN results from a combination of increased proliferation, attenuated apoptosis and defective epigenetic regulation withCALRmutations favouring apoptotic failure. The higher platelet counts reported to be seen in MPN withCALRmutations may be due to greater dysregulation of megakaryocyte apoptosis.

By array-CGH, we identified a cryptic deletion of about 3.4 Mb involving the chromosomal region 11q13.2q13.4 in a child with speech and developmental delay. Highly homologous segmental duplications related to the well-known olfactory receptor (OR)-containing clusters at 8p and 4p are located at the breakpoints of the imbalance and may be involved in its occurrence. Although these structural features are known to promote recurrent chromosomal rearrangements and previous studies had included the 11q13.2q13.4 deletion region among those considered potentially more unstable, neither deletions nor duplications of this region had been reported until now. Among the deleted genes, SHANK2 might play a role in the phenotype of the patient since it encodes a postsynaptic scaffolding protein similar to SHANK3, whose haploinsufficiency is a well-known cause of severe speech delay and autistic-like behavior, and recently deletions and mutations of SHANK2 have been described in patients with an autistic spectrum disorder or mental retardation.