INTER-LABORATORY COMPARABILITY OF QUANTITATIVE ASSESSMENT OF MUTANT NPM1: RESULTS OF THE FIRST INTERNATIONAL ROUND-ROBIN TEST PERFORMED BY THE EUROPEAN LEUKEMIA NET (ELN) MRD WORKING PARTY (WP)
Author(s): ,
Christian Thiede
Affiliations:
Medical Dept. 1,University Hospital Carl Gustav Carus,Dresden,Germany
,
Robert Baker
Affiliations:
Molecular Pathology/Genetics,Health Services Laboratories,London,United Kingdom
,
Laura Dillon
Affiliations:
Laboratory of Myeloid Malignancies,National Heart, Lung and Blood Institute,Bethesda,United States
,
Richard Dillon
Affiliations:
Department of Medical & Molecular Genetics,King's College London,London,United Kingdom
,
Nicolas Duployez
Affiliations:
Laboratory of Hematology, Biology and Pathology Center,CHU of Lille,Lille,France
,
Angela Hamblin
Affiliations:
Molecular Hematology,Oxford University NHS Foundation Trust,Oxford,United Kingdom
,
Duane Hassane
Affiliations:
Computational Biomedicine in Medicine,Weill Cornell Medicine,New York,United States
,
Michael Heuser
Affiliations:
Hematology, Hemostasis, Oncology and Stem Cell Transplantation,Hannover Medical School,Hannover,Germany
,
Sabine Jeromin
Affiliations:
Munich Leukemia Laboratory,München,Germany
,
Marta Libura
Affiliations:
Hematology Dept.,Warsaw University Hospital,Warsaw,Poland
,
Friedel Nollet
Affiliations:
Department of Laboratory Medicine,AZ Sint-Jan Brugge Oostende AV,Brugge,Belgium
,
Yishai Ofran
Affiliations:
Department of Hematology and Bone Marrow Transplantation,Technion-Israel Institute of Technology,Haifa,Israel
,
Elisabeth Opplinger Leibundgut
Affiliations:
Clinic for Hematology,Inselspital-University Hospital Bern,Bern,Switzerland
,
Heike Pfeifer
Affiliations:
Center for Internal Medicine- Medical Dept. 2,University Hospital Frankfurt,Frankfurt,Germany
,
Mikel Valganon
Affiliations:
Viapath,London,United Kingdom
,
Peter Valk
Affiliations:
Department for Hematology,Erasmus University Medical Center ,Rotterdam,Netherlands
,
Maria Teresa Voso
Affiliations:
Department of Biomedicine and Prevention,Tor Vergata University,Rome,Italy
Konstanze Döhner
Affiliations:
Department of Internal Medicine III,University Hospital of Ulm,Ulm,Germany
EHA Library. Thiede C. 06/12/20; 294376; EP457
Prof. Christian Thiede
Prof. Christian Thiede
Contributions
Abstract

Abstract: EP457

Type: e-Poster

Presentation during EHA25: All e-Poster presentations will be made available on the on-demand Virtual Congress platform as of Friday, June 12 at 08:30 CEST and will be accessible until October 15, 2020.

Background
In patients with acute myeloid leukemia (AML), assessment of measurable residual disease (MRD) has been shown to be the most powerful method for evaluating the depth of remission and the likelihood for subsequent relapse and death. High MRD-levels after end of induction have been associated with significantly decreased overall and event-free survival. MRD assessment in AML is hampered by the fact that no common marker can be used in all patients. The recently published recommendations of the ELN-AML and MRD WP recommended the use of mutant NPM1 (NPM1mut) as first priority, because this marker is present in about one third of AML patients, is stable and can be measured with high sensitivity. However, so far there have been no activities to assess the performance of laboratories and to standardize methods for quantitative NPM1mut detection based on real-time quantitative PCR (RT-qPCR).

Aims
We therefore performed a first international round robin-test involving 19 laboratories offering molecular MRD testing based on NPM1mut.

Methods
Within the ELN WP5 and WP12 we performed two consecutive round-robin tests for NPM1mut . Participating labs were asked to use their standard procedures for RT-qPCR, including RNA extraction, reverse transcription and RT-qPCR. Materials were sent out at two time points (July 2019 and December 2019). Samples (18 round 1, 21 round 2) consisted of isolated cell mixtures, RNA as well as cDNA originating from cell line dilutions (OCI-AML3/ NPM1mut A-pos in normal white blood cells (WBC) from healthy volunteer donors) covering the range from 10% down to 0.001% mutant cells. Samples containing only WBC were also distributed as negative controls. All analyzes were performed in a blinded fashion. MRD-values were reported back as NPM1mut/ABL1 in percent.

Results
Nineteen laboratories enrolled in the two rounds, participants came from 10 different countries (Germany: n=6, UK: n=4; US: n=2; Belgium, France, Israel, Italy, Netherlands, Poland, Switzerland: 1 each). All laboratories used RT-qPCR methods based on modifications of the method originally published by Gorello et al., (Leukemia 2006), and all laboratories used the ABL1-gene as reference gene for reporting. Three laboratories used commercially available assays, all other labs relied on lab-developed tests (LDTs). All particpants measured NPM1mut values with coefficients of correlation between 0.994 and 1. For the first round, all groups were able to detect the samples with the highest levels of NPM1mut, however 11% (RNA) and 16% (cDNA) failed to detect the lowest dilution step of 0.001% (false negative; FN). In contrast, false positive (FP) results were observed in 55% (RNA) respectively 52% (cDNA), usually at very low levels. Six (32%) labs were correct for all reported samples (no FN, no FP results). These data substantially improved in the second round, with only 21% (cell dilutions) and 28% (cDNA) showing either FP or FN results, 12/18 (67%) labs returning results were completely correct.

 

Conclusion
This round-robin activity indicated an overall good performance status of most laboratories in performing NPM1mut -qPCR measurements with high concordance. However, some limitations were seen with sensitivity and specificity at very high dilution levels. The improvement seen in second round strongly supports the idea to perform external validation in order to evaluate and align individual results, necessary for future clinical recommendations based on this technique.

Session topic: 03. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, Molecular markers, MRD

Abstract: EP457

Type: e-Poster

Presentation during EHA25: All e-Poster presentations will be made available on the on-demand Virtual Congress platform as of Friday, June 12 at 08:30 CEST and will be accessible until October 15, 2020.

Background
In patients with acute myeloid leukemia (AML), assessment of measurable residual disease (MRD) has been shown to be the most powerful method for evaluating the depth of remission and the likelihood for subsequent relapse and death. High MRD-levels after end of induction have been associated with significantly decreased overall and event-free survival. MRD assessment in AML is hampered by the fact that no common marker can be used in all patients. The recently published recommendations of the ELN-AML and MRD WP recommended the use of mutant NPM1 (NPM1mut) as first priority, because this marker is present in about one third of AML patients, is stable and can be measured with high sensitivity. However, so far there have been no activities to assess the performance of laboratories and to standardize methods for quantitative NPM1mut detection based on real-time quantitative PCR (RT-qPCR).

Aims
We therefore performed a first international round robin-test involving 19 laboratories offering molecular MRD testing based on NPM1mut.

Methods
Within the ELN WP5 and WP12 we performed two consecutive round-robin tests for NPM1mut . Participating labs were asked to use their standard procedures for RT-qPCR, including RNA extraction, reverse transcription and RT-qPCR. Materials were sent out at two time points (July 2019 and December 2019). Samples (18 round 1, 21 round 2) consisted of isolated cell mixtures, RNA as well as cDNA originating from cell line dilutions (OCI-AML3/ NPM1mut A-pos in normal white blood cells (WBC) from healthy volunteer donors) covering the range from 10% down to 0.001% mutant cells. Samples containing only WBC were also distributed as negative controls. All analyzes were performed in a blinded fashion. MRD-values were reported back as NPM1mut/ABL1 in percent.

Results
Nineteen laboratories enrolled in the two rounds, participants came from 10 different countries (Germany: n=6, UK: n=4; US: n=2; Belgium, France, Israel, Italy, Netherlands, Poland, Switzerland: 1 each). All laboratories used RT-qPCR methods based on modifications of the method originally published by Gorello et al., (Leukemia 2006), and all laboratories used the ABL1-gene as reference gene for reporting. Three laboratories used commercially available assays, all other labs relied on lab-developed tests (LDTs). All particpants measured NPM1mut values with coefficients of correlation between 0.994 and 1. For the first round, all groups were able to detect the samples with the highest levels of NPM1mut, however 11% (RNA) and 16% (cDNA) failed to detect the lowest dilution step of 0.001% (false negative; FN). In contrast, false positive (FP) results were observed in 55% (RNA) respectively 52% (cDNA), usually at very low levels. Six (32%) labs were correct for all reported samples (no FN, no FP results). These data substantially improved in the second round, with only 21% (cell dilutions) and 28% (cDNA) showing either FP or FN results, 12/18 (67%) labs returning results were completely correct.

 

Conclusion
This round-robin activity indicated an overall good performance status of most laboratories in performing NPM1mut -qPCR measurements with high concordance. However, some limitations were seen with sensitivity and specificity at very high dilution levels. The improvement seen in second round strongly supports the idea to perform external validation in order to evaluate and align individual results, necessary for future clinical recommendations based on this technique.

Session topic: 03. Acute myeloid leukemia - Biology & Translational Research

Keyword(s): AML, Molecular markers, MRD

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