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).
The addition of panitumumab to neoadjuvant CRT in patients with KRAS wild-type LARC resulted in a high pNC/CR rate, mostly grade 3 DC. The results of both treatment arms exceeded prespecified thresholds. The addition of panitumumab increased toxicity.
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.
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.
Aplastic anemia is associated with a variety of autoimmune syndromes. The sequence of events in our patient suggests that the hepatic failure, AFOP, and the VSAA may all have been part of an autoimmune syndrome. AFOP could be the result of immune dysregulation in this pediatric case with favorable outcome after immunosuppressive therapy and hematopoietic stem cell transplantation.
1399 Introduction: Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique and allows highly-sensitive deep-sequencing to detect molecular aberrations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Kohlmann et al., Leukemia, 2011;25:1840–8). Aims: In Phase II of the study we now established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphatic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN). 26 laboratories from 13 countries are currently part of the research consortium. Each working group selected gene targets and developed amplicons of interest to be studied in various hematological malignancies by deep-sequencing (454 Life Sciences, Branford, CT). Results: In total, 74 genes were identified by the study centers to be of interest for mutational screenings in the respective scientific working groups. Overall, 1146 primer sequences resulting in 573 amplicons were designed and tested. Where appropriate, individual genes were combined into panels and validated designs were set up as standardized preconfigured oligonucleotide primer plates. So far, in AML 679 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 864 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and the utility of this marker to detect minimal residual disease. Analyses on DNMT3A (n=126) and BCOR (n=83) were focused to perform landscape analyses and to investigate the prognostic utility of these markers. Additionally, this working group is focusing on TET2, ASXL1, and TP53 (n=195) analyses. A novel prognostic model is being developed allowing to stratify AML into prognostic subgroups based on molecular markers only. In ALL, 236 pediatric and adult cases have been screened for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R; NOTCH1, or FBXW7. Interestingly, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B3, MYD88, XPO1, FBXW7 and BIRC3. 541 cases were screened already to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance in a large unselected cohort of adult CLL. In MDS, RUNX1 mutation analyses were performed in 898 cases. Further, the prognostic relevance of TP53 mutations in MDS with isolated deletions of chromosome 5q was studied in a cohort including 105 MDS 5q- cases. Additional content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, SF1, U2AF1, ZRSR2. In BCR-ABL-negative MPN, 10 genes of interest (JAK2, MPL, EZH2, IDH1, IDH2, TET2, CBL, IKZF1, SH2B3, ASXL1) have been analyzed in a cohort of 170 cases searching for novel somatic mutations addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to 10 CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing using just 20 ng of starting DNA. A group of laboratories focused on ultra-deep sequencing analyses of the BCR-ABL tyrosine kinase domain. Analyses were performed so far on 106 cases to study the dynamics of expansion of mutated clones under various tyrosine kinase inhibitors. Conclusion: A comprehensive molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories studied a variety of hematological diseases applying standardized amplicon-based deep-sequencing assays. Distinct working groups have been forged to address scientific questions and in total 4013 cases had been analyzed thus far. Disclosures: Kohlmann: Roche Diagnostics: Honoraria; MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership; Roche Diagnostics: Research Funding.
Background Next-generation sequencing (NGS) detects somatic mutations in a high proportion of plasma cell dyscrasias (PCD), but is currently not integrated into diagnostic routine. We correlated NGS data with degree of bone marrow (BM) involvement by cytomorphology (BMC), histopathology (BMH), and multiparameter flow cytometry (MFC) in 90 PCD patients. Methods Of the 90 patients the diagnoses comprised multiple myeloma (n = 77), MGUS (n = 7), AL-amyloidosis (n = 4) or solitary plasmocytoma (n = 2). The NGS panel included eight genes CCND1, DIS3, EGR1, FAM46C (TENT5C), FGFR3, PRDM1, TP53, TRAF3, and seven hotspots in BRAF, IDH1, IDH2, IRF4, KRAS, NRAS. Results Mutations were detected in 64/90 (71%) of cases. KRAS (29%), NRAS (16%) and DIS3 (16%) were most frequently mutated. At least one mutation/sample corresponded to a higher degree of BM involvement with a mean of 11% pathologic PC by MFC (range, 0.002–62%), and ~ 50% (3–100%) as defined by both BMC and BMH. Conclusions The probability of detecting a mutation by NGS in the BM was highest in samples with > 10% clonal PC by MFC, or > 20% PC by BMC/ BMH. We propose further evaluation of these thresholds as a practical cut-off for processing of samples by NGS at initial PCD diagnosis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.