In chronic myeloid leukemia (CML), deep molecular response (DMR) is defined as molecular response 4 (MR4) or deeper. If BCR::ABL1 is undetectable the molecular response is determined by the number of control gene (CG) copies—typically ABL1 or GUSB—detected by RT-qPCR. RT-qPCR remains the gold standard for monitoring measurable residual disease (MRD). Sensitivity thresholds are set as follows: MR4 requires 10,000–31,999 ABL1 copies (or 24,000–76,999 GUSB copies, reflecting its 2.4-fold higher expression); MR4.5 requires 32,000–99,999 ABL1 copies or 77,000-239,999 GUSB copies; and MR5 requires ≥100,000 ABL1 or ≥240,000 GUSB copies. The efficiency of reverse transcriptase (RT) enzymes used during cDNA synthesis can significantly impact transcript quantification and thereby MR classification. As part of the European Treatment and Outcome Study for CML 2024 (EUTOS 2024) initiative, this study aimed to evaluate the influence of various RT enzymes on assay sensitivity and MR classification—an important consideration for treatment discontinuation decisions, as outlined in the European LeukemiaNet (ELN) 2025 guidelines. Samples from 12 CML patients in deep molecular response (DMR) — 7 with major and 5 with atypical BCR::ABL1 transcripts — were analyzed. Equal amounts of RNA, within the range recommended by the respective manufacturers, were reverse-transcribed using seven different reverse transcriptase (RT) enzymes: SuperScript II (SSII), GoScript, MMLV, High Capacity, VILO, Clara, and GoTaq. Quantification of ABL1, GUSB, and BCR::ABL1 transcripts was performed using standardized RT-qPCR protocols. Therefore, the only variable in this analysis was the choice of RT enzyme. Using SSII, the expected GUSB/ABL1 expression ratio was confirmed (mean 2.57, SD 0.35; range 2.12–3.16), yielding consistent MR classification: 1 sample at MR4.5, 11 at MR5 for both CGs. High Capacity produced comparable results in 10 samples, with 2 classified at GUSB-MR5 but ABL1-MR4.5 (mean ratio 3.17, SD 0.53). MMLV showed slightly higher GUSB expression (mean ratio 3.42, SD 1.15) resulting in the classification of 5 samples as GUSB-MR5 but ABL1-MR4.5, while 6 remained at MR5 for both CGs and 1 sample at MR4.5 for both CGs. VILO and GoTaq exhibited markedly higher GUSB/ABL1 ratios—10.64 (SD 2.71) and 7.08 (SD 1.49), respectively—causing inconsistent MR classification across most samples. GoScript produced similar ABL1 and GUSB levels (mean ratio 1.3, SD 0.18) but overall lower transcript yields, leading to lower sensitivity. Clara showed highly variable performance and inconsistent GUSB/ABL1 ratios (SD 3.19, range 1.31-10.5), with 6/11 samples yielding false-negative BCR::ABL1 results. These findings indicate that ABL1 copy numbers are often insufficient, reflecting a lower analytical sensitivity. GUSB also showed suboptimal performance in most cases, with the exception of VILO, which tended to overestimate GUSB expression. This study highlights the critical impact of reverse transcriptase choice on MR classification in CML, particularly in cases of undetectable BCR::ABL1. Only SuperScript II, High Capacity, and MMLV produced consistent and ELN-compliant results, including assessment of DMR. In contrast, GoTaq and GoScript generally produced insufficient copy numbers of both ABL1 and GUSB, resulting in reduced analytical sensitivity. VILO also yielded low ABL1 copy numbers but tended to slightly overestimate GUSB expression. Clara, due to inconsistent control gene transcription and a high risk of false-negative MRD results, appears unsuitable for clinical use in this context. We recommend that testing laboratories should validate their cDNA synthesis protocols to ensure that measured CG copy numbers fall within ELN-defined MR ranges, especially when evaluating patients for treatment-free remission.Support: EUTOS 2024, MH CZ – DRO (IHBT, 00023736)

Background. Approximately 98% of CML patients express e13a2 and/or e14a2 BCR::ABL1 mRNA isoforms resulting from genomic breakpoints that fall within a ~2.9 Kb region in BCR and a ~140 Kb region in ABL1. Rarer isoforms result from breakpoints in other parts of BCR and/or ABL1.Additional BCR::ABL1-associated abnormalities have also been described that may be prognostically relevant. We set out to assess the ability of long read nanopore sequencing to detect genomic BCR::ABL1 fusions and any BCR::ABL1-associated rearrangements or other structural variants in patients with CML. Methods. DNA from patients with CML (n=30) were fragmented to 5-10 Kb and prepared for sequencing using the Oxford Nanopore Technologies (ONT) ligation sequencing kit v14. Adaptive sampling was used to target a panel of 240 genes recurrently involved in hematological malignancies in all 30 cases (37.1 Mb total), with 11kb of padding sequence at either end of each gene to maximise coverage. The full lengths of chr9 and chr22 (223.9 Mb total) was also included for 25/30 cases. Sequencing was performed on the MinION Mk1b, Dorado (ONT) and minimap2 were used for basecalling and alignment, SV were called with sniffles2. Selected rearrangements were confirmed by targeted PCR and sequencing. BCR::ABL1 transcript type was known for all cases and full karyotype for 18 cases. Results. Median on-target sequencing depth was 38X, representing a 7-fold enrichment over non-targeted sequence. Median read N50 was 7.8 Kb. Evidence for BCR::ABL1 was called by sniffles2 in 27/30 cases. In the remaining 3 cases, the fusion call was initially filtered in 2 samples due to a low fraction of fusion reads (0.16 and 0.17), and only 2 fusion supporting reads in 1 sample. Evidence for the fusion was found upon manual review. BCR::ABL1 transcript type was concordant with the genomic breakpoints in all cases, including 3 with atypical fusions (e14a2, n=14; e13a2, n=10; e14a2/e13a2, n=3; e1a2, n=2; e14a3, n=1). Cytogenetic results were available for 18/30 cases and a Ph chromosome was seen in 12/18 of these, including 11 where it was seen as the sole abnormality. Two of these 11 cases (cases 15 and 30) had unexpected additional rearrangements detected by nanopore sequencing: case 15 had a t(1;9)(q25.3;q34.12) with a 6.1 Kb inversion at chr9:130,709,000 within 1Kb of the t(9;22) reciprocal breakpoint. Case 30 had an additional t(3;22)(p21.31;q11.23). One patient (case 26) had a Ph chromosome plus complex additional chromosome abnormalities that were partially resolved by nanopore sequencing. A normal karyotype was seen in 1/18 cases, with ABL1 inserted into BCR as determined by FISH. A 2.17 Mb deletion between chr9:130,842,023-133,012,328 was detected by nanopore sequencing, with breakpoints for BCR::ABL1 and the reciprocal fusion found at either end. Complex rearrangements involving 9q34 and/or 22q11 were detected in 5/18 samples by cytogenetics. Genomic breakpoints were detected by nanopore sequencing in all 5 cases, 2 of which were found to have additional complexity. Case 1 had a 46,XY,t(9;22;9)(q34;q11;q13) by karyotyping, but nanopore suggested 2 distinct t(9;22) events, with the additional t(9;22) revealed to have breakpoints at 9q21 and 9q34, separated by a 58.5 Mb duplication. Case 3 had a 46,XY,t(3;9)(p14;q34) by cytogenetics but nanopore sequencing indicated a t(3;9)(p21.31;q34.12), t(3;9)(p21.2;q34.13) and an additional t(3;22)(22q11.23;p21.2). Of the 12 cases that did not have cytogenetic results, 2 had evidence for additional rearrangements. Case 11 had a t(7;9)(q21.2;q34.12) and t(7;22)(q21.2;q11.23) whereas case 17 had a 154 bp inversion close to the BCR::ABL1 junction and a reciprocal breakpoint approximately 1 Mb downstream of BCR in intron 19 of CABIN1. Conclusions. This study demonstrates nanopore sequencing is able to successfully detect typical and atypical BCR::ABL1 genomic breakpoints and can revealthe underlying complexity in samples with BCR::ABL1-associated rearrangements, as well as other structural variants. Further work is required to determine the clinical significance of this additional genomic complexity in CML.

Abstract The initial rate of reduction of BCR::ABL1 mRNA on TKI therapy, usually measured as the halving time over the first 3 months of treatment, has prognostic value for both early and late responses as well as the likelihood of achieving TFR. Standardized measurement of pretreatment disease levels by RT-qPCR, however, is problematic due to the different properties of the 3 established reference genes (ABL1, GUSB, BCR) used for CML MRD analysis. These differences are not relevant for standard MRD assessments, but the use of ABL1 or BCR results in reference gene-specific distortions at high disease burdens that limit their utility for assessment of baseline disease levels. GUSB is completely independent of BCR::ABL1 and is thus theoretically the best reference gene to determine halving times. To date, however, no comparative analysis of the utility of different reference genes to assess early response kinetics has been performed. We have evaluated the utility of halving times derived using GUSB and ABL1 as reference genes in FASCINATION, a multicenter, prospective, open-label, interventional phase II trial designed to evaluate the efficacy and tolerability of asciminib following <6 weeks treatment with other TKIs or <4 weeks of hydroxyurea. Halving times in days were calculated by comparing BCR::ABL1/ABL1 and BCR::ABL1/GUSB levels at trial entry (63% and 23%, respectively, at the start of asciminib) to the corresponding measurements at 3 months on asciminib. Halving times and IS levels at 3 months were compared to molecular outcomes at 18 months. Of 103 cases with available data, 45 achieved DMR at 18 months (MR4 or better; median 0.0015% IS) and 58 did not achieve DMR (median 0.052% IS). Median halving times were shorter for patients who achieved DMR using both ABL1 (13.5 vs 20.9, P<0.001) and GUSB (11.2 vs 18.1, P<0.001) as reference genes. Similarly, IS values at 3 months were significantly lower for cases who achieved DMR at 18 months (0.23% vs 1.6%; P<0.001). Using ROC analysis, the predictive value of GUSB halving times (AUC GUSB = 0.792) for achievement of DMR at 18 months was superior to both ABL1 halving times (AUC ABL1 = 0.746) and IS values at 3 months (AUC = 0.776). Similarly, we found that GUSB halving times correlated better (r=0.58, Spearman’s rank correlation) with month 18 IS levels considered as a continuous variable compared to both ABL1 halving time (r=0.47) and month 3 IS levels (r=0.51). Focusing on GUSB, 19/51 (37%) cases had a halving time shorter than the median for all cases (13.9 days) but were not in DMR at 18 months. 12 of these 19 cases also had lower than the median IS levels at 3 months (0.73%). Conversely 13/52 cases (25%) had a long halving time but achieved DMR at 18 months. 8/13 also had higher than median IS levels at 3 months. Of the 45 cases who achieved DMR at 18 months, 11 (24%) had IS levels at 3 months that were higher (median = 3.97%, range 0.77%-35.3%) than the median for all cases (0.73%). Only 1/11 had a shorter than median GUSB halving time. Conversely, 17/58 (29%) of cases who were not in DMR at 18 months had <0.73% IS at 3 months (median = 0.40; range 0.05-0.70); only 5/17 had long GUSB halving times. These data indicate that combining halving times with 3 month IS data adds limited additional predictive value. Since GUSB measurements remain largely unstandardized, we assessed the performance of the AcroMetrix™ BCR-ABL Panel (Thermo Fisher), recently calibrated to GUSB and BCR as well as ABL1, to standardize MRD results using GUSB as a reference gene in two centers. Established GUSB conversion factors (CF) derived by sample exchange were essentially indistinguishable from those derived using the panel [Lab 1: existing CF (0.99) / panel CF (1.02) = 0.97; Lab 2: existing CF (1.57) / panel CF (1.40) = 1.12, thus validating the panel to standardize GUSB measurements. We conclude that halving times over the first 3 months of asciminib treatment using GUSB or ABL1 as well as IS levels at 3 months are all predictive of molecular response at 18 months, but the predictive value of GUSB halving times are greatest. We therefore recommend that standardized baseline BCR::ABL1/GUSB measurements are incorporated into future studies to enable comprehensive assessment of the value of BCR::ABL1 halving times for routine management.

Diverse haematological neoplasms are driven by tyrosine kinase (TK) fusion genes formed by recurrent or non-recurrent genomic rearrangements. The resulting chimeric proteins often present excellent targets for treatment with kinase inhibitors, and the fusion transcripts or genomic junctions can be used as specific targets for molecular monitoring. Whilst the TK genes involved are generally well characterised (e.g. ABL1, PDGFRA, FGFR1), the fusion partners are very diverse, presenting a challenge for detection and characterisation of these structural variants (SV) using current diagnostic methods. We assessed the ability of targeted nanopore sequencing using adaptive sampling to detect fusion genes in myeloid neoplasms. We sequenced genomic DNA from patients (n = 20) with a known or suspected TK gene fusion and identified rearrangements in 18 cases, including all cases with a known TK fusion, typical and atypical BCR::ABL1 rearrangements, an 843Kb deletion causing a FIP1L1::PDGFRA fusion, novel AGAP2::PDGFRB and NFIA::PDGFRB fusions, and a complex CCDC88C::PDGFRB rearrangement with multiple translocation events. The approach was fast (<72 h/sample from DNA to result), flexible with minimal hands-on laboratory time, and provided accurate, patient-specific characterisation of genomic breakpoints.

INTRODUCTION:Patient decision aids (PtDA) complement shared decision-making with healthcare professionals and improve decision quality. However, PtDA often lack theoretical underpinning. We are codesigning a PtDA to help people with increased genetic cancer risks manage choices. The aim of an innovative workshop described here was to engage with the people who will use the PtDA regarding the theoretical underpinning and logic model outlining our hypothesis of how the PtDA would lead to more informed decision-making. METHODS:Short presentations about psychological and behavioural theories by an expert were interspersed with facilitated, small-group discussions led by patients. Patients were asked what is important to them when they make health decisions, what theoretical constructs are most meaningful and how this should be applied to codesign of a PtDA. An artist created a visual summary. Notes from patient discussions and the artwork were analysed using reflexive thematic analysis. RESULTS:The overarching theme was: It's personal. Contextual factors important for decision-making were varied and changed over time. There was no one 'best fit' theory to target support needs in a PtDA, suggesting an inductive, flexible framework approach to programme theory would be most effective. The PtDA logic model was revised based on patient feedback. CONCLUSION:Meaningful codesign of PtDA including discussions about the theoretical mechanisms through which they support decision-making has the potential to lead to improved patient care through understanding the intricately personal nature of health decisions, and tailoring content and format for holistic care. PATIENT CONTRIBUTION:Patients with lived experience were involved in codesign and coproduction of this workshop and analysis as partners and coauthors. Patient discussions were the primary data source. Facilitators provided a semi-structured guide, but they did not influence the patient discussions or provide clinical advice. The premise of this workshop was to prioritise the importance of patient lived experience: to listen, learn, then reflect together to understand and propose ideas to improve patient care through codesign of a PtDA.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories ( n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB , respectively, during the study period and 98% of laboratories were able to detect MR 4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1 IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

Introduction: we previously reported the outcome of the DESTINY study, in which patients with CML in chronic phase in at least MMR, were invited to stop treatment only if they remained in at least MMR after a 12 month period of 50% de-escalation of their TKI. Recurrence was defined as confirmed loss of MMR. The 3 year probabilities of recurrence-free survival (RFS) were 72% and 36% in patients who entered the study in MR4 (or deeper) and MMR respectively. We also observed that RFS was better in patients in whom RT-qPCR (IS) ratios did not rise between months 0-12. We now report on one of the secondary outcomes, namely a comparison of standard RT-qPCR and digital droplet (dd) PCR in the prediction of molecular recurrence. Methods: the study completed recruitment in April 2015 and LPLV was in April 2018. Further follow-up to identify late molecular recurrence occurred in 2021. 157/174 patients discontinued their TKI at month 12, of whom 22 had a RT-qPCR ratio consistent with MMR but not MR4 at month 12. A further 7 patients were lost to follow-up for a variety of reasons. As our hypothesis was that ddPCR might offer enhanced sensitivity over RT-qPCR, we compared samples from patients who stopped their TKI in at least MR4 as defined by RT-qPCR (n=128), and investigated the relationship between the level of response at stopping and subsequent molecular recurrence. ddPCR was used to determine the number of ABL1 and BCR::ABL1 copies for each patient. BCR::ABL1/ABL1 values were calculated from total copy numbers across n=3 technical replicates for each target and results were converted to the International Scale (IS). 2µL cDNA was used per replicate, and primers and probes were per the EAC design previously described (Gabert et al, 2003). Results: Of 22 patients who had RT-qPCR ratios between 0.01% IS and 0.1% IS (MMR) at month 12, 19 (86%) had molecular recurrence: 5/22 had RT-qPCR ratios consistent with MMR at study entry (month 0) and 2 had recurrence but the remainder experienced increasing RT-qPCR levels over the period of de-escalation and all had molecular recurrence. RT-qPCR results were available for all 128 patients in MR4 or better at month 12, but ddPCR results for only 117/128 due to technical failure (n=3) or insufficient residual sample for ddPCR analysis (n=8). There was in general correlation of results between the methodologies but incomplete concordance with ddPCR ratios >RT-qPCR, ddPCR = RT-qPCR and ddPCR < RT-qPCR in 54, 14 and 49 samples respectively. The absolute values for the differences were generally small (median increases of 0.0018 when ddPCR>RT-qPCR and 0.0007 when RT-qPCR>ddPCR) (Table 1). The probabilities of molecular recurrence at 5 years by RT-qPCR defined response levels at study entry of MR4 (n=30), MR4.5 (n=42) and MR5 (n=56) were 50%, 43% and 17% respectively (p<0.001). The equivalent probabilities by ddPCR defined MR3/MR4 (n=36), MR4.5 (n=25) and MR5 (n=56) were 58%, 32% and 21% respectively (p<0.001) (Figure 1). ddPCR reclassified 10 patients in MR4 by RT-qPCR to MMR and 5 of these 10 had recurrence, equivalent to the recurrence rate of ddPCR defined MR4 but considerably lower than RT-qPCR defined MMR. 56 patients were in MR5 by RT-qPCR, of whom 20 were reclassified by ddPCR as MR4.5 (n=11), MR4 (n=6) or MMR (n=3), of whom 5 had recurrence (2 x MR4, 3 x MR4.5). Similarly 56 patients were in MR5 by ddPCR and 20 were reclassified as MR4.5 (n=14) or MR4 (n=6), of whom 6 had recurrence (5 x MR4.5). Further analyses using receiver operator curves were unable to identify PCR ratios by either method that had sufficient specificity to be used in clinical practice. Conclusions: Patients in MR5 are at a low risk of disease recurrence. The risk of recurrence was highest in patients in MMR at the time of stopping and for this group, the value of a methodology offering higher sensitivity than standard RT-qPCR is questionable. For patients in deeper response levels, even those in MR5, we could not identify any advantage of ddPCR over RT-qPCR as performed in our laboratories. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Purpose Approximately 1 – 2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients. Methods BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR). Results In total, 330 blood samples (2–34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios. Conclusions Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.

Background Fetal structural anomalies, which are detected by ultrasonography, have a range of genetic causes, including chromosomal aneuploidy, copy number variations (CNVs; which are detectable by chromosomal microarrays), and pathogenic sequence variants in developmental genes. Testing for aneuploidy and CNVs is routine during the investigation of fetal structural anomalies, but there is little information on the clinical usefulness of genome-wide next-generation sequencing in the prenatal setting. We therefore aimed to evaluate the proportion of fetuses with structural abnormalities that had identifiable variants in genes associated with developmental disorders when assessed with whole-exome sequencing (WES). Methods In this prospective cohort study, two groups in Birmingham and London recruited patients from 34 fetal medicine units in England and Scotland. We used whole-exome sequencing (WES) to evaluate the presence of genetic variants in developmental disorder genes (diagnostic genetic variants) in a cohort of fetuses with structural anomalies and samples from their parents, after exclusion of aneuploidy and large CNVs. Women were eligible for inclusion if they were undergoing invasive testing for identified nuchal translucency or structural anomalies in their fetus, as detected by ultrasound after 11 weeks of gestation. The partners of these women also had to consent to participate. Sequencing results were interpreted with a targeted virtual gene panel for developmental disorders that comprised 1628 genes. Genetic results related to fetal structural anomaly phenotypes were then validated and reported postnatally. The primary endpoint, which was assessed in all fetuses, was the detection of diagnostic genetic variants considered to have caused the fetal developmental anomaly. Findings The cohort was recruited between Oct 22, 2014, and June 29, 2017, and clinical data were collected until March 31, 2018. After exclusion of fetuses with aneuploidy and CNVs, 610 fetuses with structural anomalies and 1202 matched parental samples (analysed as 596 fetus-parental trios, including two sets of twins, and 14 fetus-parent dyads) were analysed by WES. After bioinformatic filtering and prioritisation according to allele frequency and effect on protein and inheritance pattern, 321 genetic variants (representing 255 potential diagnoses) were selected as potentially pathogenic genetic variants (diagnostic genetic variants), and these variants were reviewed by a multidisciplinary clinical review panel. A diagnostic genetic variant was identified in 52 (8.5%; 95% CI 6.4-11.0) of 610 fetuses assessed and an additional 24 (3.9%) fetuses had a variant of uncertain significance that had potential clinical usefulness. Detection of diagnostic genetic variants enabled us to distinguish between syndromic and non-syndromic fetal anomalies (eg, congenital heart disease only vs a syndrome with congenital heart disease and learning disability). Diagnostic genetic variants were present in 22 (15.4%) of 143 fetuses with multisystem anomalies (ie, more than one fetal structural anomaly), nine (11.1%) of 81 fetuses with cardiac anomalies, and ten (15.4%) of 65 fetuses with skeletal anomalies; these phenotypes were most commonly associated with diagnostic variants. However, diagnostic genetic variants were least common in fetuses with isolated increased nuchal translucency (>= 4.0 mm) in the first trimester (in three [3.2%] of 93 fetuses). Interpretation WES facilitates genetic diagnosis of fetal structural anomalies, which enables more accurate predictions of fetal prognosis and risk of recurrence in future pregnancies. However, the overall detection of diagnostic genetic variants in a prospectively ascertained cohort with a broad range of fetal structural anomalies is lower than that suggested by previous smaller-scale studies of fewer phenotypes. WES improved the identification of genetic disorders in fetuses with structural abnormalities; however, before clinical implementation, careful consideration should be given to case selection to maximise clinical usefulness. Funding UK Department of Health and Social Care and The Wellcome Trust. Copyright (c) 2019 The Author(s). Published by Elsevier Ltd.

(Abstracted from Lancet 2019;393:747–757) Fetal structural anomalies, detected by ultrasonography, have a range of genetic causes, including chromosomal aneuploidy, copy number variations (CNVs), and pathogenic sequence variants in developmental genes. Conventional prenatal cytogenetic analysis was historically the first-line method to investigate these anomalies, but chromosomal microarray analysis has been adopted more recently, as this test is able to detect smaller, but significant, CNVs.

For patients with chronic myeloid leukaemia (CML), treatment guidelines recommend monitoring response to treatment with tyrosine kinase inhibitors (TKIs) by testing the BCR-ABL1 fusion gene transcript level using reverse transcriptase quantitative polymerase chain reaction. Despite recent efforts to standardise protocols for BCR-ABL1 testing, some variability remains among laboratories in the UK regarding the techniques used and the approach to reporting results. This increases the risk of misinterpretation of results by both clinicians and patients. An expert panel met to discuss current issues surrounding BCR-ABL1 testing in the UK and to develop guidance for laboratories, with emphasis on the optimal approach to reporting laboratory results. Topics included the minimum required information to include in the laboratory report, units of measurement, test sensitivity and BCR-ABL1 transcript variants. To aid communication between laboratories and clinics, standard forms were generated that could be used by (i) clinics when submitting samples to laboratories, and (ii) laboratories when reporting results to clinics. Standardising the way in which BCR-ABL1 test results are reported from laboratories to clinics should help to improve communication, interpretation of results and patient care.

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.

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2(V617F)-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10(-10)) and rs2201862 (MECOM; meta-analysis P=1.96 × 10(-9)). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2(V617F)-positive cases. rs9376092 has a stronger effect in JAK2(V617F)-negative cases with CALR and/or MPL mutations (Breslow-Day P=4.5 × 10(-7)), whereas in JAK2(V617F)-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ(2) P=7.3 × 10(-7)). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype.