European Research Initiative on CLL (ERIC) guidelines for the detection of minimal residual disease in CLL using multi- parameter (≥4CLR) flow cytometry
Andy C. Rawstron; Claudia Fazi; Andreas Agathangelidis; Neus Villamor; Remi Letestu; Josep Nomdedeu; Carlos Palacio; Olga Stehlikova; Karl- Anton Kreuzer; Stuart Liptrot; David O’Brien; Ruth M. de Tute; Martin Spacek; Johan Dobber; Arnon P. Kater; Peter Gambell; Asha Soosapilla; Gerard Lozanski; Gabriele Brachtl; Ke Lin, Curtis Hanson; Jeffrey L. Jorgensen; Maryalice Stetler-Stevenson; Constance Yuan; Beth Broome; Fiona Craig; Julio Delgado; Carol Moreno; Francesc Bosch; Alexander Egle; Michael Doubek; Sarka Pospisilova; Stephen Mulligan; David Westerman; Tait Shanafelt; Andy Pettitt; Thomas J. Kipps; William G. Wierda; Florence Cymbalista; Michael Hallek; Peter Hillmen; Emili Montserrat and Paolo Ghia on behalf of the ERIC consortium
Lessons from the several years of attempting to harmonise CLL MRD by flow cytometry
• The CLL community needs to progress – Flow MRD can easily achieve 10-5, but do we want this if it then means there are insufficient cells left to test the novel assay that can achieve 10-6 ? • The participating labs need flexibility – Variable equipment specification, reagents restricted to certain suppliers, compatibility with local procedures, locally critical markers (CD160, ROR1) • Researchers and biotech need to know that their assay meets requirements without re-validating every individual combination – Provide a specification for the required components so that developers can demonstrate approved standard. Guidelines on the validation of cell-based assays • Sensitivity – Either: “lowest signal detectable above background” – Or: “true positive / true positive + false negative” • Limit of Blank (LOB) = highest signal in the absence of measurand, calculated as mean (blank) + 1.645 SD (95% of negative values are below this limit) • Limit of Detection (LOD) = level at which 95% of samples with low level of measurand are detected above the limit of blank, calculated as LOB + 1.645 SD • Limit of Quantification (LoQ) = lowest level of measurand that can be reliably detected and whose total error (bias + Imprecision) meets a desired criterion for accuracy (clinical utility)
Cytometry Part B (Clinical Cytometry) 84B: 315-323 Clinically appropriate level of variation: When BCR-ABL RQ-PCR accepted as a trial end-point, 95% limit of agreement = ± 5-fold
CML: best 95% LOA +/- 2-fold using synthetic standards CLL: Target: ±3-fold (0.5log), preferably ±2-fold (0.3 log) Multiparameter flow CLL MRD Guidelines: aims of the project
• The primary aim of this project was to identify and validate the specification of an assay that could provide a stable core set of markers for MRD detection: – at the levels required by the IWCLL guidelines or better – compatible with published outcome data – independent of instrument/reagent manufacturer – offer sufficient flexibility for the use and validation of additional markers. • The secondary aim of this study was to compare the assay with High Throughput Sequencing to determine the most appropriate application of each technology. 4 vs. 6 vs. 8-CLR Component markers
PerCP PE- APC- FITC PE -Cy5.5 Cy7 APC H7 CD45 CD3 CD19 CD5 CD20 CD38 CD19 CD5 CD81 CD22 CD19 CD5 CD43 CD79b CD19 CD5
CD3 CD38 CD5 CD19 CD79b CD20 CD81 CD22 CD5 CD19 CD43 CD20
4CLR 20-50 CLL events in ≥2 of 3 tubes 6CLR 20-50 CLL events in both tubes
PerCP- V450 V500 FITC PE Cy5.5 PE-Cy7 APC APC-H7 CD5 CD3 CD81 CD79b CD22 CD19 CD43 CD20 1-tube 8-CLR dilution analysis
10
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CLR assay CLR -
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tube 8 tube -
0.01
0.001
0.0001 CLL % leucocytes using 1 using % CLL leucocytes
0.00001 0.00001 0.0001 0.001 0.01 0.1 1 10 Expected CLL % leucocytes
A) 8-CLR 1-tube panel dilution analysis PerCP- V450 V500 FITC PE Cy5.5 PE-Cy7 APC APC-H7 CD5 CD3 CD81 CD79b CD22 CD19 CD43 CD20 6-markers are sufficient for MRD detection
10 1
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Below LOD Above LOQ CLR assay CLR
- 0.4 0.1
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tube 8 tube -
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0.0001 CLL % leucocytes using 1 using % CLL leucocytes
-0.8
core marker single tube assays (log transformed) assays tube single marker core Difference in CLL in Difference of %8 leucocytes between 0.00001 -1 0.00001 0.0001 0.001 0.01 0.1 1 10 0.0001 0.001 0.01 0.1 1 10 100 Expected CLL % leucocytes CLL % of leucocytes average of 8-CLR and core marker single tube assays
A) 8-CLR 1-tube panel dilution B) 6 markers are sufficient for analysis detection of MRD PerCP- V450 V500 FITC PE Cy5.5 PE-Cy7 APC APC-H7 CD5 CD3 CD81 CD79b CD22 CD19 CD43 CD20 1-tube 6-CLR analysis limit of detection 10-5 with acceptable inter-operator variation
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core panel core 0.1 -
0.01
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CLL % leucocytes using MRD using % CLL leucocytes 0.0001
0.00001 0.00001 0.0001 0.001 0.01 0.1 1 10 Expected CLL % leucocytes
A) 6-CLR 1-tube core marker panel B) Variation tested in 19 students at dilution analysis: LoD 0.001% ESCCA-sponsored educational meetings Figure 3: A platform-independent specification for MRD-flow reagents Acceptable Sub-optimal
CLL cells Normal B-cells Normal T-cells A platform-independent specification for MRD-flow reagents
Control populations in normal Minimum Typical peripheral blood Expression Relative Antigen fluorescence (% pos vs. intensity ‡ Positive Negative control) (preferred)
CD5 Positive (>20%) CD3+ T-cells CD19+ B-cells >30 (>65)
CD20 Weak CD19+ B-cells CD3+ T-cells >13 (>20) CD43 Positive (>20%) CD3+ T-cells CD20+ B-cells >15 (>40) CD79b Weak CD20+ B-cells CD3+ T-cells >15 (>30) CD81 Weak CD3+ T-cells Granulocytes >12 (>20) LoD: (≤) 1 in a million
Case 1 Case 2 Case 3
Sequence 1 Sequence 2 Average of S1 & S2
Dilutional analysis (Milan) assessing high-throughput sequencing: analysis of 3 CLL cases diluted into leucocytes from leucodpletion filters in serial 1:10. Each CLL clone was tagged with 2 sequences, 1 productive and 1 non-productive. The plot shows the CLL sequence as a percentage of nucleated genomes. For log-transformed data above the LoQ, linearity = 1.15, correlation co-efficient (Pearson R) = 0.980, average difference = 0.15 log, 95% limit of agreement 0.76 log. ClonoSEQ HTS in comparison to Sanger sequencing and MRD-flow in patient samples
• Identifying a clonal marker: HTS MRD-core (CLL ClonoSEQ REF cells % of (neoplastic detected a dominant clone (median leucocytes) sequences % of 99.1%, range 7.8-100%) in 66/68 H24O286M <0.001 0.00035 samples vs. 57/68 by Sanger H003O335M <0.001 0.00054 sequencing H235O273M <0.001 0.00061 – 1 technical issue / 1 LC-MBL H78O277M [0.002] 0.0022 H93O275M 0.008 0.0039 • Clonal IGH CDR3 sequences H69O280M 0.012 0.0051 were identical in 49/57 H112O101M <0.001 0.0082 H204O278M 0.46 0.46 – 5 cases Sanger-identified clone H104O351M 23 0.55 detected by HTS (3/5 H104O351M 13 0.55 unproductive, 2/5 not dominant) H67O274M 1.48 0.56 – 3 discrepant in LC-MBL (HTS H64O385M 1.59 0.99 most likely correct) H130O285M 0.3 10.5 • Repeat analysis confirms dominant 12/13 concordant at 0.01% threshold clone in 10/10 CLL/HC-MBL cases (1 flow below LoQ) 3/13 MRD detected by ClonoSEQ not by flow HTS shows greater variation in quantification above 0.01% but better accuracy below 0.01%
4-CLR vs. ≥6-CLR flow cytometry HTS vs. ≥6-CLR flow cytometry
Comparison of the MRD-core panel with ClonoSEQ high throughput sequencing: analysis of 3 CLL cases diluted into leucocytes from leucodpletion filters in serial 1:10 dilutions and 13 CLL cases after treatment. The results are not corrected for LoD/LoQ. For log-transformed data above the LoQ, linearity = 0.905, correlation co- efficient (Pearson R) = 0.870, average difference = 0.078 log, 95% limit of agreement 1.5 log.
Multiparameter flow CLL MRD guidelines: aims of the project • Identify assay specification for CLL MRD analysis to achieve at least 0.01% limit of quantification – CD19 (or appropriate gating markers) in combination with CD5, CD20, CD43, CD79b, CD81 markers meeting defined specification – Interoperator variation in analysis: 95% LOA within 2-fold (log- scale, ±0.3) achieved by operators after reviewing protocol / test cases. • Compare ≥4CLR MRD flow with high-throughput sequencing – Optimal combination direct quantitative assay to 0.01 - 0.001% (10-4 to 10-5) combined with HTS to exclude disease below 1 in a million (10-6). Different ERIC-harmonised approaches for MRD detecetion
B-cell Requires capacity to reflex to full enumeration + Relatively inexpensive and simple. MRD assay if CLL cells <1% clonality &/or B-cells polyclonal. assessment Published outcome data Limit of detection >0.005% 4-colour 4-tube Does not require pre-treatment More material required to phenotype for typical CLL achieve higher detection limits Harmonised with published Intermediate LoD/LoQ outcome data Intermediate amount of 6-colour 2-tube Does not require pre-treatment materialrequired to achieve phenotype for typical CLL higher detection limits. Flexibility for individual laboratory 6-colour core requirements Knowledge of pre-treatment panel for ≥6- LoD 0.001% (10-5), LoQ 0.0025% phenotype preferable. colour assays Allows simultaneous analysis of additional markers LoD 0.0001% (10-6) Further development work on High throughput Objective analysis, does not standardisation of the sequencing necessarily require expert quantification interpretation Acknowledgements Johan Dobber, Arnon Kater Academic Medical Center, Amsterdam, The Netherlands. Remi Letestu, Florence Cymbalista AP-HP, Hôpital Avicenne, Bobigny, France Martin Spacek Charles University in Prague, Czech Republic Neus Villamor, Julio Delgado Hospital Clínic, Barcelona, Spain. Josep Nomdedeu, Carol Moreno Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. Claudia Fazi, Paolo Ghia Università Vita-Salute San Raffaele, Milan, Italy Gabriele Brachtl, Alexander Egle Paracelsus Medical University, Salzburg , Austria. Stuart Liptrot, David O'Brien St. James's Hospital, Dublin, Ireland. Andy C. Rawstron, Ruth M. de Tute, Peter Hillmen St. James's Institute of Oncology, Leeds Teaching Hospitals, UK. Olga Stehlikova, Michael Doubek, Sarka Pospisilova University Hospital Brno, Czech Republic. Karl-Anton Kreuzer, Michael Hallek University of Cologne, Köln, Germany Carlos Palacio, Francesc Bosch Vall d'Hebron Hospital, Barcelona, Spain. Peter Gambell, David Westerman Peter MacCallum Cancer Centre, East Melbourne, Australia Asha Soosapilla, Stephen Mulligan Royal North Shore Hospital, University of Sydney, Australia Constance Yuan, Maryalice Stetler-Stevenson Center for Cancer Research, National Institutes of Health, Bethesda, MD, USA. Gerard Lozanski Department of Pathology, The Ohio State University Curtis Hanson, Tait Shanafelt Mayo Clinic, Rochester, Minnesota, USA. Jeffrey L. Jorgensen, William G. Wierda MD Anderson Cancer Center, University of Texas Beth Broome, Thomas J. Kipps Moores Cancer Center, University of California, La Jolla, CA, USA. Fiona Craig University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Carol Moreno, EMILI MONTSERRAT and PAOLO GHIA HTS vs. Sanger: Andreas Agathangelidis Dilution studies: Claudia Fazi
Ruth de Tute, ANDREW JACK and PETER HILLMEN
Frans Nauwelaers, Lucia Testolin, Jingyi Chen and Noel Warner. BD Biosciences provided custom and commercial conjugates for testing
Michael Wenger for meeting support
Comparison of flow vs. next generation sequencing Which patients can be monitored: diagnosis of CLL • No universal molecular abnormality • WHO Definition: CLL cells usually co-express CD5 and CD23 • WHO Immunophenotype: using flow cytometry, the tumour cells express dim surface IgM/IgD, CD20, CD22, CD5, CD19, CD79a, CD23, CD43 and CD11c (weak). CD10 is negative and FMC& and CD79b are usually negative or weakly expressed in typical CLL. Some cases may have an atypical immunophenotype (e.g. CD5- or CD23-, FMC7+ or CD11c+, strong sIg, or CD79b+). • iwCLL/NCI guidelines: CLL cells co-express the T-cell antigen CD5 and B-cell surface antigens CD19, CD20, and CD23. The levels of surface immunoglobulin, CD20, and CD79b are characteristically low compared with those found on normal B cells. Each clone of leukemia cells is restricted to expression of either kappa or lambda immunoglobulin light chains. Variations of the intensity of expression of these markers may exist and do not prevent inclusion of a patient in clinical trials for CLL. Difficult to validate diagnostic panels – circular argument for the gold standard CLL Diagnostic Panel
• An invitation to participate in this study was circulated among members of the ERIC and ESCCA scientific groups. • Responses were received from 158 members of which 154 were actively involved in CLL diagnosis: 100/154 (65%) were from diagnostic laboratories, 14/154 (9.1%) were CLL clinicians and 36/154 (23%) worked in both laboratory and clinic. • The diagnostic workload was >20 cases per week in 23/154 (15%), 5-20 per week in 82/154 (53%) and <5% in 49/154 (32%). • Responders were invited to classify 35 antibodies selected from publications on the diagnosis of B-lymphoproliferative disorders as being required, recommended, suggested, uninformative, or of unknown value for the diagnosis of CLL. >75% of respondents marker is required for CLL diagnosis:
>50% of respondents marker is recommended for CLL diagnosis:
Proposal: required (minimum) and recommended panel for diagnosis • Marker panel required for diagnosis: – CD19 / CD5 / CD23 / CD20 / Kappa / Lambda
• Additional markers recommended for diagnosis and required for clinical trials: – CD43 / CD79b / CD81* / CD22 / CD10
• Frequently recommended but not essential for diagnosis and monitoring: – CD45 / CD38 / CD200 / (FMC7)
• Present in current diagnostic criteria but not universally recommended: – IgM/D and CD11c:
• * CD81 was considered as a required or recommended by only 40% of participants but this marker is an essential component for the consensus MRD monitoring panels [REFS] and therefore recommended by the steering committee for clinical trials. Figure 3a: A platform-independent specification for MRD-flow reagents Acceptable Sub-optimal
CLL cells Normal B-cells Normal T-cells Minimum Typical Control Population in normal Are the same Relative Expression peripheral blood Antigen criteria required fluorescence (% pos vs. for atypical CLL? intensity control) ‡ Positive Negative (preferred) CD19 Positive (>95%) Yes CD20+ B-cells CD3+ T-cells >20† CD16/56+ NK- CD5 Positive (>20%) Yes CD3+ T-cells >14 (>18) cells CD20+CD27- CD20+27+ CD23 Positive (>20%) ? Not required >5† Naïve B-cells Memory B-cells CD20 Weak Not required CD19+ B-cells CD3+ T-cells >5 (>20) Igκ Weak & restricted Restricted to either CD20+ B-cells CD3+ T-cells >10† Igλ to either Igκ or Igλ Igκ or Igλ CD43 Positive (>20%) Not required CD3+ T-cells CD20+ B-cells >7 (>50)
CD79b Weak Not required CD20+ B-cells CD3+ T-cells >11 (>30)
CD81 Weak Not required CD3+ T-cells Granulocytes >5 (>8)
CD22 Weak Not required CD20+ B-cells CD3+ T-cells >10† CD20+27+ CD10 Negative (<20%) Yes Granulocytes >10† Memory B-cells Definition of weak: median fluorescence intensity at least 20% lower than normal peripheral blood B-cells, range to be determined within each laboratory (ICSH/ISLH/CLIA guidelines for stability require <20% variation, therefore reduction in fluorescence intensity less than 20% may reflect antigen/sample stability) † consensus, not specifically validated