SummaryMolecular 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.
Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21 500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6–85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.
EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)–polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18–21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.
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).
2812 Since the discovery of the JAK2-V617F mutation, the relative level of the mutant allele has been widely studied to gain further insights into the biology of myeloproliferative neoplasms and to establish utility in predicting clinical outcome. Mutant allele burden has been correlated with risk of thrombotic events in essential thrombocythemia, severity of the disease phenotype in polycythemia vera (PV) and survival in primary myelofibrosis (PMF). JAK2-V617F has also been quantified to assess disease response, with significant reductions in allele burden reported in PV patients following interferon (IFN) α-2b and pegylated IFN α-2a treatment. Moreover, serial DNA-based quantitative polymerase chain reaction (Q-PCR) assays have been used after allogeneic transplantation for myelofibrosis to predict outcome and guide donor lymphocyte infusion. With quantification of JAK2-V617F being considered an endpoint in a number of trials with novel agents including JAK2 inhibitors, it is important that assays are robust, mutant-specific and afford a suitable level of sensitivity. However, numerous JAK2-V617F Q-PCR assays have now been published, which based on experience of molecular monitoring in other hematological malignancies, are likely to vary markedly in their performance. Indeed, this concern was confirmed by the results of a quality control (QC) exercise involving 5 European LeukemiaNet (ELN) laboratories, each with their own established JAK2-V617F Q-PCR assay, who reported markedly differing levels of mutant allele percentage (ranging from 23–80%) in a QC sample distributed by UK NEQAS that comprised a 1 in 30 dilution of HEL cells (which harbor multiple copies of JAK2-V617F and lack the wild type [WT] JAK2 allele) in K562 cells (WT JAK2). To address this issue, a joint initiative was established within European LeukemiaNet between the Chronic Myeloproliferative Diseases and Minimal Residual Disease working groups (WP9 & WP12) to systematically evaluate a range of published and “in-house” JAK2-V617F assays, with the aim of identifying sensitive mutant-specific assays that perform similarly on different Q-PCR platforms – factors of fundamental importance for reliable tracking of disease response. The study involved 12 expert centers - in 6 European countries and included 1 US laboratory, which between them used Q-PCR platforms from 3 different manufacturers. The study involved central distribution of DNA from serial dilutions of JAK2-V617F mutant cell lines (HEL &/or UKE-1) in K562 and plasmid standards for JAK2 WT, JAK2-V617F and independent control genes (albumin, cyclophilin). Reagents for each assay were coded and also centrally distributed; reaction conditions for each coded assay were provided. Laboratories with established published or in-house Q-PCR assays were encouraged to perform these in parallel with the coded test assays on the distributed QC materials, to control for local variations in test conditions. Data from each QC round were submitted by participating laboratories for independent central analysis (Dept. of Genetics, King's College London); conclusions were drawn concerning the performance of the various assays before breaking the code to reveal their identities. Overall, 7 successive QC rounds were conducted, evaluating 9 assays (6 published, 3 unpublished “in-house”). Six assays were eliminated, due to insufficient/inferior sensitivity (n=5) or inconsistent performance (n=1). Of the 3 assays with better performance profile, the assay published by Lippert et al (Blood 2006;108 :1865) reliably quantified JAK2-V617F across the diagnostic range, with consistent results obtained between laboratories irrespective of Q-PCR platform. The 2 remaining assays (Larsen et al, Br J Haematol. 2007;136 :745 & in-house assay from the Oppliger Leibundgut lab [Bern]), afforded better sensitivity, capable of detecting a 0.08–0.008 % level of JAK2-V617F in DNA preparations from HEL and K562 cells. The latter assays were tested on serial post-transplant samples from 2 patients allografted for JAK2-V617F mutant PMF, successfully identifying the presence of residual disease, complementing chimerism analysis to predict clinical outcome. In conclusion, this ELN study has identified robust assays suitable for quantifying JAK2-V617F in clinical trials and which merit further investigation as a tool to guide management of patients undergoing allogeneic transplant. Disclosures: No relevant conflicts of interest to declare.
e20678 Background: The clinical significance of common epidermal growth factor receptor (EGFR) mutations (exon 19 deletions and exon 21 L858 mutations) in non-small cell lung cancer is well known1. However, 2-8% of NSCLC patients harbour an uncommon EGFR mutation2. Prognosis and response to tyrosine kinase inhibitors (TKIs) is unclear in this subgroup. The most frequent uncommon EGFR mutations are exon 18 G719 and exon 21 L861. Although not frequently reported, these appear to have favourable outcomes with TKIs3. Methods: We conducted a retrospective review of all patients with uncommon EGFR mutations identified at laboratories in 4 UK centres, between 2012 and 2016. Patient’s not treated with a TKI were excluded. Techniques including cold PCR were in use at contributing laboratories. The T790M resistance mutation was excluded from our study. For patients with uncommon EGFR mutations, patient characteristics and response to TKIs were analysed. Results: 29 patients with uncommon EGFR mutations, with both rare point mutations (86%) and deletions (14%), were identified. All patients were treated with a TKI. Age ranged from 37 to 85 (median 67). The majority (86%) of patients were performance status 1. TKIs used were afatinib (31%), gefitinib (24%) and erlotinib (45%). 19 patients (66%) benefitted from TKI treatment (38% partial response (PR), 28% stable disease (SD). 10 patients (34%) did not respond, including all four with exon 20 mutations. 15 mutations were exon 18 G719 mutations: 40% had a PR and 40% stable disease with TKI therapy. There were two L861 mutations, both responded with a PR. 13 patients remained on TKI treatment at data cut off. The median progression free survival at this time was 8.1months. Tabulated details of response according to mutation will be presented. Conclusions: The majority (66%) of NSCLC patients with uncommon EGFR mutations benefitted from TKI therapy in this series. These high clinical benefit rates, typified by patients with G719 mutation, are in line with the limited previous reports 1,3. Exon 20 mutations were associated with TKI resistance, in line with the literature4.
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