Analytical Validation of Patient-Specific PCR-Based MRD Assessment for Use As a Primary Endpoint in CLL Clinical Trials
Authors: Schilhabel Anke , Knecht Henrik , Langerak Anton W. , van Dongen Jacques J.M. , Kneba Michael , Ray Jill , Punnoose Elizabeth , Kim Doris , Haberberger Thomas , Bernaards Coen , Zhu Yuda , Lewin-Koh Sock-Cheng , Ritgen Matthias 
Published: October 2019

Abstract
Introduction. Minimal residual disease (MRD) is an objective measure of disease status defined by the number of leukemic cells in the blood or bone marrow of leukemic patients. In recent clinical studies of chronic lymphocytic leukemia (CLL), undetectable MRD levels (< 1 tumor cell/10,000 leukocytes) have been shown to correlate with prolonged progression free survival (PFS) and overall survival, independent of treatment or known risk factors. MRD assessment has been proposed as an alternative to PFS as a primary endpoint in frontline CLL pivotal studies to evaluate the efficacy of novel therapies at an earlier time-point.
Thorough standardization and validation are needed to use MRD as a primary surrogate endpoint. Allele specific oligonucleotide (ASO)-PCR of immunoglobulin (IG) gene rearrangements is a method for quantifying MRD using patient-specific PCR primers and has been standardized by the EuroMRD Consortium (www.EuroMRD.org). Given that each patient has individualized PCR primers designed for their leukemic clone, this posed a unique challenge for the analytical validation studies to demonstrate that the assays are uniform in their reproducibility and analytical sensitivity to measure MRD across patients with CLL. Here we report a comprehensive, IVD-guided analytical validation of the ASO-PCR technique according to the guidance of regulatory authorities. We provide evidence that the ASO-PCR methodology can reproducibly measure MRD to the required threshold of 10-4, across patients with CLL.
Results. Performance of ASO-PCR was assessed using a combination of retrospective data from the CLL11 clinical trial and prospectively performed experiments. Patient assays from 60 CLL patients were tested in two EuroMRD laboratories to demonstrate linearity across the measurement range of 10-1 to 10-5, and a limit of detection of 6.3x10-5, which is below the cut-off of 10-4 used for defining MRD negativity. Concordance of the method to an orthogonal method was determined from the previously published comparison of flow cytometry with ASO-PCR (Boettcher et al., Leukemia 2009; 23: 2007) with 93.8 % overall agreement between both methods (n=452). Agreement of MRD status was >97% when comparing individually designed ASO primers for the same patient within the lab. The overall agreement between the two different laboratories using independently designed ASO-PCR assays was 93.5%.
Precision was assessed above and below the threshold of 10-4 using ASO-PCR assays of 3 individual patient samples diluted to appropriate MRD levels listed in Table 1. Theexperiment was designed to mimic sources of variation by evaluating MRD samples over the course of the clinical study (3 days x 2 operators x 3 patients x 2 laboratories x 3 replicates). Overall variability was estimated using a mixed effects model including fixed patient effects and random effects for operator and day. Based on the known MRD distribution of frontline CLL patients, we estimate acceptable overall variability on the order of 80% CV at lower concentrations (≤ 3.2x10-4) and 40% CV at higher concentrations (> 3.2x10-4). This precision estimate provides reasonable misclassification rates (< 5%) due to the fact that the majority of patients had MRD levels either well above or below 10-4 level. Experiments also addressed stability of patient specimens and critical assay components.
Table 1. Precision of ASO-PCR results obtained at MRD levels 10-2 to 10-5Table 1.Estimated Total CV (%) Averaged Across 3 patientsMRD levelKielErasmus1.00E-028.7034.791.00E-0310.0235.743.20E-0415.8233.271.00E-0431.7036.383.20E-0589.8978.801.00E-05258.06277.27
Conclusion. The analytical validation studies described here provide evidence that the ASO-PCR methodology, standardized by EuroMRD, performs well to reproducibly detect MRD status across CLL patients at the threshold of 10-4. These studies serve as an example for the validation of personalized, patient-specific quantitative clinical assays for use as a primary endpoint in clinical trials.
The authors would like to acknowledge the valuable work of the following people who contributed to this work: M. Brüggemann (UKSH Kiel), R Raja, C. Cox, W. Darbonne, R. Desai, and K. Trunzer.

Disclosures
Langerak: DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam). van Dongen:BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics.; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge.Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.. Royalty income for EuroClonality-BIOMED-2 Consortium; Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences)., Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL.. Ray:Genentech, Inc.: Employment. Punnoose:Genentech, Inc.: Employment. Kim:Genentech, Inc.: Employment. Haberberger:Genentech, Inc.: Employment. Bernaards:Roche: Employment. Zhu:Genentech, Inc.: Employment. Lewin-Koh:Genentech, Inc.: Employment. Ritgen:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.