Consensus on BCR-ABL1 reporting in chronic myeloid leukaemia in the UK.

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.

Bacteriophage SPP1 tail tube protein self-assembles into ß-structure-rich tubes.

The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the ß-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its ß-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable ß-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40-56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations.

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

Capsid structure and its stability at the late stages of bacteriophage SPP1 assembly.

The structure of the bacteriophage SPP1 capsid was determined at subnanometer resolution by cryo-electron microscopy and single-particle analysis. The icosahedral capsid is composed of the major capsid protein gp13 and the auxiliary protein gp12, which are organized in a T=7 lattice. DNA is arranged in layers with a distance of ~24.5 Å. gp12 forms spikes that are anchored at the center of gp13 hexamers. In a gp12-deficient mutant, the centers of hexamers are closed by loops of gp13 coming together to protect the SPP1 genome from the outside environment. The HK97-like fold was used to build a pseudoatomic model of gp13. Its structural organization remains unchanged upon tail binding and following DNA release. gp13 exhibits enhanced thermostability in the DNA-filled capsid. A remarkable convergence between the thermostability of the capsid and those of the other virion components was found, revealing that the overall architecture of the SPP1 infectious particle coevolved toward high robustness.

Establishment and validation of analytical reference panels for the standardization of quantitative BCR-ABL1 measurements on the international scale.

BACKGROUND: Current guidelines for managing Philadelphia-positive chronic myeloid leukemia include monitoring the expression of the BCR-ABL1 (breakpoint cluster region/c-abl oncogene 1, non-receptor tyrosine kinase) fusion gene by quantitative reverse-transcription PCR (RT-qPCR). Our goal was to establish and validate reference panels to mitigate the interlaboratory imprecision of quantitative BCR-ABL1 measurements and to facilitate global standardization on the international scale (IS). METHODS: Four-level secondary reference panels were manufactured under controlled and validated processes with synthetic Armored RNA Quant molecules (Asuragen) calibrated to reference standards from the WHO and the NIST. Performance was evaluated in IS reference laboratories and with non-IS-standardized RT-qPCR methods. RESULTS: For most methods, percent ratios for BCR-ABL1 e13a2 and e14a2 relative to ABL1 or BCR were robust at 4 different levels and linear over 3 logarithms, from 10% to 0.01% on the IS. The intraassay and interassay imprecision was <2-fold overall. Performance was stable across 3 consecutive lots, in multiple laboratories, and over a period of 18 months to date. International field trials demonstrated the commutability of the reagents and their accurate alignment to the IS within the intra- and interlaboratory imprecision of IS-standardized methods. CONCLUSIONS: The synthetic calibrator panels are robust, reproducibly manufactured, analytically calibrated to the WHO primary standards, and compatible with most BCR-ABL1 RT-qPCR assay designs. The broad availability of secondary reference reagents will further facilitate interlaboratory comparative studies and independent quality assessment programs, which are of paramount importance for worldwide standardization of BCR-ABL1 monitoring results and the optimization of current and new therapeutic approaches for chronic myeloid leukemia.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Fast-mode duplex qPCR for BCR-ABL1 molecular monitoring: innovation, automation, and harmonization.

Reverse transcription quantitative polymerase chain reaction (RTqPCR)is currently the most sensitive tool available for the routine monitoring of disease level in patients undergoing treatment for BCRABL1 associated malignancies. Considerable effort has been invested at both the local and international levels to standardise the methodology and reporting criteria used to assess this critical metric. In an effort to accommodate the demands of increasing sample throughput and greater standardization, we adapted the current best-practice guidelines to encompass automation platforms and improved multiplex RT-qPCR technology.

Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy.

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.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study.

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 MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS 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.

Guidelines for the measurement of BCR-ABL1 transcripts in chronic myeloid leukaemia.

Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Assessment of individual molecular response in chronic myeloid leukemia patients with atypical BCR-ABL1 fusion transcripts: recommendations by the EUTOS cooperative network.

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.

Frequent upregulation of MYC in plasma cell leukemia.

Plasma cell leukemia (PCL) is a rare form of monoclonal gammopathy, which can originate de novo or evolve from multiple myeloma (MM) as a terminal leukemic phase. Previous cytogenetic studies of PCL have reported the presence of complex karyotypes with involvement of multiple unidentified chromosomal regions. We report here the analysis of 12 PCL (10 primary and two secondary) by metaphase and FISH analysis combined with oligonucleotide array data (244 k, Agilent). Interphase-FISH results were compared with those from a series of 861 newly diagnosed patients with MM. Cytogenetic analysis was successful on 11 patients, all of whom showed clonal chromosomal abnormalities. Compared with MM, t(11;14)(q13;q32) (42% versus 15%; P = 0.027) and t(14;16)(q32;q23) (25% versus 4%; P = 0.010) were more frequent in PCL, although neither the specific partner chromosome involved in the IgH translocation nor the ploidy status predicted for survival. Chromosomes 1, 8, 13, and 16 showed the highest number of copy number alterations with 8q24 being the chromosomal region most frequently involved. In eight of 12 patients we found abnormalities (translocations, one amplification, small deletions, and duplications) that directly targeted or were very close to MYC. Only four of these changes were detected by routine FISH analysis using commercial probes with the others exclusively detected by arrays. Quantitative reverse transcription polymerase chain reaction demonstrated that these different abnormalities were associated with increased levels of MYC mRNA. We conclude that MYC dysregulation by complex mechanisms is one of the major molecular events in the oncogenesis of PCL.

Rapid identification of JAK2 exon 12 mutations using high resolution melting analysis.

Diverse JAK2 exon 12 mutations have been described in patients with V617F-negative polycythemia vera. Development of a sensitive detection assay capable of identifying any of these mutations is required for medium-throughput diagnostic screens. Non-mutated and mutant JAK2 exon 12 alleles were amplified from patient samples and cloned into plasmid vectors, then used to determine the sensitivity of a novel high-resolution melting-curve assay designed to detect all mutant JAK2 exon 12 alleles tested. High resolution melting analysis was more sensitive than direct sequencing and capable of detecting exon 12 mutations in granulocytes at moderate levels. In a blinded analysis of DNAs from V617F-negative erythrocytosis patients, with direct sequencing and allele-specific PCR used in one laboratory and high resolution melting analysis in another, high resolution melting successfully identified JAK2 exon 12 mutations in all 4 mutation-positive patients. High resolution melting analysis is a rapid, sensitive and high-throughput technique that is suitable for screening for JAK2 exon 12 mutations.

Lengsin is a survivor of an ancient family of class I glutamine synthetases re-engineered by evolution for a role in the vertebrate lens.

Lengsin is a major protein of the vertebrate eye lens. It belongs to the hitherto purely prokaryotic GS I branch of the glutamine synthetase (GS) superfamily, but has no enzyme activity. Like the taxon-specific crystallins, Lengsin is the result of the recruitment of an ancient enzyme to a noncatalytic role in the vertebrate lens. Cryo-EM and modeling studies of Lengsin show a dodecamer structure with important similarities and differences with prokaryotic GS I structures. GS homology regions of Lengsin are well conserved, but the N-terminal domain shows evidence of dynamic evolutionary changes. Compared with birds and fish, most mammals have an additional exon corresponding to part of the N-terminal domain; however, in human, this is a nonfunctional pseudoexon. Genes related to Lengsin are also present in the sea urchin, suggesting that this branch of the GS I family, supplanted by GS II enzymes in vertebrates, has an ancient role in metazoans.

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26.

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.

Influence of the MDM2 single nucleotide polymorphism SNP309 on tumour development in BRCA1 mutation carriers.

BACKGROUND: The MDM2 gene encodes a negative regulator of the p53 tumour suppressor protein. A single nucleotide polymorphism (SNP) in the MDM2 promoter (a T to G exchange at nucleotide 309) has been reported to produce accelerated tumour formation in individuals with inherited p53 mutations. We have investigated the effect of the MDM2 SNP309 on clinical outcome in a cohort of patients with germline mutations of BRCA1. METHODS: Genomic DNA was obtained for 102 healthy controls and 116 patients with established pathogenic mutations of BRCA1 and Pyrosequencing technologytrade mark was used to determine the genotype at the MDM2 SNP309 locus. RESULTS: The polymorphism was present in 52.9% of the controls (G/T in 37.3% and G/G in 15.6%) and 58.6% of the BRCA1 mutation carriers (47.4% G/T and 11.2% G/G). Incidence of malignancy in female BRCA1 carriers was not significantly higher in SNP309 carriers than in wildtype (T/T) individuals (72.7% vs. 75.6%, p = 1.00). Mean age of diagnosis of first breast cancer was 41.2 years in the SNP309 G/G genotype carriers, 38.6 years in those with the SNP309 G/T genotype and 39.0 years in wildtype subjects (p = 0.80). CONCLUSION: We found no evidence that the MDM2 SNP309 accelerates tumour development in carriers of known pathogenic germline mutations of BRCA1.

Recognition and separation of single particles with size variation by statistical analysis of their images.

Macromolecules may occupy conformations with structural differences that cannot be resolved biochemically. The separation of mixed molecular populations is a pressing problem in single-particle analysis. Until recently, the task of distinguishing small structural variations was intractable, but developments in cryo-electron microscopy hardware and software now make it possible to address this problem. We have developed a general strategy for recognizing and separating structures of variable size from cryo-electron micrographs of single particles. The method uses a combination of statistical analysis and projection matching to multiple models. Identification of size variations by multivariate statistical analysis was used to do an initial separation of the data and generate starting models by angular reconstitution. Refinement was performed using alternate projection matching to models and angular reconstitution of the separated subsets. The approach has been successful at intermediate resolution, taking it within range of resolving secondary structure elements of proteins. Analysis of simulated and real data sets is used to illustrate the problems encountered and possible solutions. The strategy developed was used to resolve the structures of two forms of a small heat shock protein (Hsp26) that vary slightly in diameter and subunit packing.

Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing.

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.