International Standardized Approach to Molecular and Flow Cytometric Residual Disease Monitoring in CLL.
Authors: Rawstron A. C. , Villamor N. , Zehnder J. L. , Ghia P. , Ritgen M. , Bottcher S. , Lozanski G. , Mone A. P. , Colomer D. , Moreno C. , Geuna M. , Evans P. A. , Kneba M. , Natkunam Y. , Coutre S. E. , Caligaris-Cappio F. , Hallek M. J. , Byrd J. C. , Montserrat E. , Hillmen P. 
Published: October 2019

Abstract
Conventional therapy in CLL is not curative, partly because minimal residual disease (MRD) is usually detectable after therapy. Recent therapeutic approaches achieve higher complete remission (CR) rates, and often aim for MRD eradication as this has repeatedly been shown to predict improved outcome. However, many different MRD techniques have been used to assess response, making it extremely difficult to interpret and compare different clinical trials. The aim of this international collaboration is to develop standardized flow cytometric and PCR approaches to MRD monitoring in CLL that are broadly applicable; to assess their relative sensitivity and specificity in a variety of laboratories; and to establish a standardized reporting convention to facilitate the interpretation of clinical trials. These techniques may be used as a benchmark for assessing response and comparing the efficacy of different therapeutic approaches. PCR approaches using consensus primers to the immunoglobulin heavy chain (IgH) gene are straightforward and frequently used, but have highly variable sensitivity between patients. In the proposed standardized method, the IgH gene is amplified using the BIOMED-2 primers (van Dongen et al, Leukemia 2003, 17:2257) and an allele-specific CDR3 primer is designed. CLL cells are identified by RQ-PCR using the allele-specific oligonucleotide (ASO) primer coupled with intronic JH primers and consensus reporter probes. The amount of PCR product is compared to a single copy housekeeping gene (albumin) to identify the number of CLL cells in the sample. This approach reproducibly quantitates as few as 1 CLL cell in 10,000 other leucocytes, and may provide qualitative information below this level. Multi-parameter flow cytometric analysis with combinations such as CD19/CD5/CD79b/CD20 is reported to have a sensitivity approaching that of ASO-PCR. However, such approaches are less effective in situations where normal B-cells lack CD20, e.g. in patients treated with combinations including rituximab and in normal bone marrow. To circumvent these problems, the group has tested 58 individual four-colour combinations of antibodies identified from protein expression profiling and previously published studies. CD79b/CD43/CD19/CD5, CD81/CD22/CD19/CD5, and CD20/CD38/CD19/CD5 provided the least inter-laboratory variation, independent of methodology. They show low contamination by non-CLL cells and were therefore selected as a core panel for MRD analysis. Dilutional studies indicate that a quantitative sensitivity equivalent for detection of 1 CLL cell in 10,000 leucocytes is achievable; the practical sensitivity is being tested in blinded dilutional analyses. The proposed flow cytometry approach is applicable to all sample types and all therapeutic regimes, and sufficiently rapid and sensitive to guide therapy to an MRD negative status in real time. The PCR approach is highly sensitive but more complex, and is most suited to retrospective response assessment of clinical trials. We have identified the appropriate standardized analytical methods for both techniques and are currently validating these findings. These approaches will allow direct comparison of efficacy between different clinical trials, allowing more rapid international progress towards identifying a curative approach.