Summary Measurement of BCR activator of RhoGEF and GTPase ‐ABL proto‐oncogene 1, non‐receptor tyrosine kinase (BCR‐ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large‐scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR‐ABL1‐positive samples with levels ranging from molecular response (MR)1·0–MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR‐ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR‐ABL1 in all samples to MR4·0. Detection rates for deep‐response samples were 95·7% at MR4·5, 78·3% at MR4·7 and 87·0% at MR5·0. Interlaboratory coefficient of variation was indirectly proportional to BCR‐ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment‐free remission.

Abstract Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.

Summary Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.

We report a sibling-pair and a 4-year old child from two families with an atypical presentation in Osteogenesis imperfecta (OI). In the sib-pair, the older sibling initially came to medical attention due to a fracture history (Patient 1) and she was shown to have a COL1A2 mutation. In addition, she also had developmental delay, facial dysmorphism, and a history of frequent infections which led to a search for an alternate diagnosis. ArrayCGH revealed a 4.3 Mb duplication on chromosome 19q13.42q13.43, which was confirmed by FISH analysis. On further familial analysis, the younger sibling who had no previous fracture history was also found to have the COL1A2 mutation and tested positive for the 19q13.42q13.43 duplication (Patient 2). The 19q13 duplication appears to be the cause of intellectual disability in these siblings but given that this is a chromosomal duplication, it is still possible that there is an as yet unidentified cause that may account for the combined phenotype in this family. Patient 3 was a 4-year old child presenting with a femoral fracture, blue sclerae, developmental delay, and joint hypermobility. Genetic analyses confirmed a COL1A2 mutation but also revealed an 8.8 Mb deletion of 11q24.2q25, confirmed by G-band chromosome analysis. We discuss the differing phenotypes in patients presenting with atypical OI and stress the need to consider ancillary investigations in individuals presenting with heterogeneous phenotypic symptoms, not entirely attributable to OI.

The European LeukaemiaNet (ELN) measurable residual disease (MRD) working group have published consensus guidelines to standardise molecular genetic MRD testing of the t(8;21)(q22;q22.1) RUNX1::RUNX1T1, inv(16)(p13.1q22) CBFB::MYH11, t(15;17)(q24.1;q21.2) PML::RARA and NPM1 type A markers. A study featuring 29 international laboratories was performed to assess interlaboratory variation of testing, and the subsequent interpretation of results, both crucial to patient safety. The majority of participants in this study were able to detect, accurately quantify and interpret MRD testing results correctly, with a level of proficiency expected from a clinical trial or standard of care setting. However, a number of testing and interpretive errors were identified that in a patient setting would have led to misclassification of patient outcomes and inappropriate treatment pathways being followed. Of note, a high proportion of participants reported false positive results in the NPM1 marker negative sample. False positive results may have consequences clinically, committing patients to unneeded additional chemotherapy and/or transplant with the attendant risk of morbidity and mortality, and therefore highlights the need for ongoing external quality assessment (EQA)/proficiency testing (PT) in this area. Most errors identified in the study were related to the interpretation of results. It was noted that the ELN guidance lacks clarity for certain clinical scenarios and highlights the requirement for urgent revision of the guidelines to elucidate these issues, and related educational efforts around the revisions to ensure effective dissemination.

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AimsHaematological malignancies represent a diverse group of diseases with complex diagnostic requirements. National Institute for Health and Care Excellence (NICE) Haematological Cancer: Improving Outcomes Guidance was published in 2003 and updated in 2016 (NG47), providing recommendations for service delivery including Specialist Integrated Haematological Malignancy Diagnostic Services (SIHMDSs). This survey assessed the implementation of NG47 guidelines, with a specific focus on implementation in relation to laboratory SIHMDS delivery.MethodsA survey was issued to the 17 SIHMDSs identified in England. The questionnaire covered laboratory configuration, information systems, integrated reporting and multidisciplinary team (MDT) working recommendations.ResultsIn the 10 responding SIHMDS, full implementation of recommendations was not achieved. Higher levels of implementation were reported in ‘colocated’ services compared with ‘networked’ SIHMDS. Increased guideline implementation was reported with longer duration since initial establishment of a SIHMDS and for laboratory based as opposed to clinical (MDT) reporting recommendations.ConclusionsOur survey highlights variable implementation of NICE guidance across SIHMDS, with likely inequity of access, standardisation and quality in haemato-oncology diagnostics. Provision of a more structured framework for guideline implementation could assist in increasing compliance to meet the goals of quality and equity of access to harmonised haemato-oncology diagnostics across the NHS in England. This would provide a basis for evaluating the clinical benefits and health economic impact of the SIHMDS model on patient care and outcomes.