MRD Evaluation The Austrian experience Gabriele Brachtl Molekularzytologisches Labor III. Medical Department of Hematology, Oncology, Hemostaseology, Rheumatology and Infectiology Head: Univ. Prof. Dr. Richard Greil University Hospital Salzburg www.onkologie-salzburg.com
Mabtenance MRD measurements of ~180 patients Austria, Slovakia, Bulgaria, Romania 18 x peripheral blood 3 x bone marrow 3780 MRD samples
MRD Analysis in Salzburg
Minimal Residual Disease International standardized MRD flow assay Leeds Protocol (Leukemia, 2007): multi parameter flow cytometry Technical requirements 10 ml Fresh to one day old material (high quality sample necessary) At least 10 ml heparinzied blood Lysis: ammonium chloride lysis, pooled (needs to be as complete as possible without losing any other cells) At least 2,5 mio stained cells, at least 500.000 leucocytes acquired EXTENSIVE data analysis: strictly following SOPs EXPERIENCED flow cytometry operators VERY RESTRICED SOPs
MRD: 4 color 5 tubes EXTENSIVE setup procedure: adapt protocols to equipment, simulation of MRD, detection limit requirements flow cytometer: FC-500 (5-color, Beckman Coulter) used different fluorochromes used some different antibodies (Coulter) FITC PE PC5 PC7 clonality of CLL cells Quality control MRD analysis (1) kappa light chain lambda light chain CD5 CD19 (2) CD45 CD14 CD3 CD19 (3) CD20 CD38 CD5 CD19 (4) CD81 CD22 CD5 CD19 (5) CD43 CD79b CD5 CD19
Sample processing in Salzburg day -1 Notification by FAX on day before sample arrival Buffer preparation day 0 Check sample: correctly sent? correct sample? labeling? White blood cell count Sample preparation and staining (lysis, cell count, staining) Flow cytometric measurement Documentation (total about 2½ hours mostly hands on) later Pooled data analysis (about 20-30 min/sample)
MRD detection according to Leeds protocol FITC PE PC5 PC7 clonality of CLL cells (1) kappa light chain lambda light chain CD5 CD19 Quality control (2) CD45 CD14 CD3 CD19 (3) CD20 CD38 CD5 CD19 MRD analysis (4) CD81 CD22 CD5 CD19 (5) CD43 CD79b CD5 CD19 MRD detection 3 stainings Total of 6 additional markers to the CD19/CD5 expression Each combination of markers helps discriminate CLL cells from T cells, B cells, and B progenitors
CD5 CD5 CD79b III. Medical Department of Hematology, Oncology, Detection of MRD positive CLL samples MRD positive > 0.01% CLL cells of leucocytes More than 50 CLL cells for 500.000 acquired leucocytes MRD positive patient sample = MRD positive at least in 2 out of 3 stains Stain 3 Stain 4 Stain 5 CD38 CD81 CD43
CD5 CD5 CD79b CD5 CD22 CD5 CD5 CD38 CD5 III. Medical Department of Hematology, Oncology, MRD positive patient sample total leucocytes (CD45+): 409.094 Stain 3 #CLL = 115 0,028 % CD20 CD20 CD38 Stain 4 #CLL = 110 0,027% CD22 CD81 CD81 Stain 5 #CLL = 119 0,029 % CD79b CD43 CD43
MABTENANCE Overview of first MRD samples
Mabtenance MRD samples Patients n = 30 Austria n = 23 Slovakia n = 7 Total MRD samples n = 98 5 patients are already at end of cycle 4 6 pre-screening patient samples: before start of initial treatment
MRD status of first patients at Screening PB N = 25 BM N = 23 Peripheral Screening Blood - PB Samples 20 15 15 10 10 5 0 MRD NEG MRD POS Peripheral Blood (PB) vs. Bone Marrow (BM) n = 23 n.s. 3/23: MRD positive in BM but MRD negative in PB 1/23: MRD negative in BM but MRD positive in PB (PB MRD neg in consecutive time points)
% CLL cells of leucocytes III. Medical Department of Hematology, Oncology, MRD over time: after 3rd cycle 5 patients Pat A: from just MRD pos (0,02%) to clearly positive (0,47%) Rest: B-E MRD neg (D,E: normal B cells coming up after end of cycle 1) 4 out of 5 MRD negative 0,50 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 SC-BM SC-PB C1 C2 C3 C4 Pat A Pat B Pat C Pat D Pat E 0,01%
LOG % CLL cells of leucocytes III. Medical Department of Hematology, Oncology, MRD over time: LOG scale 1,0000 SC-BM SC-PB C1 C2 C3 C4 0,1000 0,0100 0,0010 Pat A Pat B Pat C Pat D Pat E 0,0001
Technical limitations and controls of MRD analysis Contamination rate = percentage of CD19+CD3+ cells events = limit below which it is technically not possible to enumerate CLL cells CD3 There are no CD19+/CD3+ cells! CD19 5 CD19+/CD3+ events in 500.000 leucocytes 0,001%
Technical limitations and controls of MRD analysis Contamination rate = percentage of CD19+CD3+ cells events = limit below which it is technically not possible to enumerate CLL cells CD3 There are no CD19+/CD3+ cells! CD19 Original Leeds Leukemia 2007 n = 141 77 Contamination rate median % of leucocytes 5 95th percentile 0,007% 0.0006 0.028% Salzburg MRD 0,001% 0,000-0,036% Below 0,01% 78% (110/141) 90% (69/77)
SS SS SS SS III. Medical Department of Hematology, Oncology, Technical limitations and controls of MRD analysis Total number of events required Detection limit 0,01% 50 CLL cells in 500.000 leucocytes 98% 88% Leucocyte numbers: Important to calculate actual number of leucocytes in sample and percent of CLL cells CD45 37% 3% CD45 Pooled lysis: equal erythrocyte lysis in all tubes equal percent of CD45+ Ideally all acquired events should be CD45+ & Quality of blood sample CD45 CD45
Technical limitations and controls of MRD analysis Total number of events required Detection limit 0,01% 50 CLL cells in 500.000 leucocytes Original Leeds Leukemia 2007 n = 141 77 Median # of acquired cells/leucocytes Salzburg MRD 325.050 457.875 range 43.519 592.353 21.450 547.910
MABTENANCE 8 color MRD Assay
LOG % CLL cells of leucocytes 8 color assay III. Medical Department of Hematology, Oncology, 8 color assay 4 color assay Comparison: Results of 4 vs. 8 color MRD assay R² = 0.99 p < 0.0001 parallel data sets n = 72 4 color assay LOG % CLL cells of leucocytes
8 color assay 4 color assay Comparison: Results of 4 vs. 8 color MRD assay n.s. 49 - MRD neg n = 72 22 - MRD pos 1 - discrepancy 4 color 8 color BM sample MRD pos 0,0121% (2 out of 3) MRD neg 0,0074% PB and C1-PB: MRD neg
8 color assay Advantages of a 8 color assay Less time consuming Less time needed for sample preparation, measurements and analysis (only 2 instead of 5 tubes) Cheaper Less antibodies and reagents needed All parameters in one tube More efficient analysis, higher impact of result Better controls Contamination control in same tube
MABTENANCE First steps taken
First steps taken MRD assay setup 4 color assay 8 color assay Start MRD RQ-PCR for Salzburg patients
What s missing Pre-initial-therapy blood sample Individual immunophenotyp especially important for atypical CLL patients Very important for 8 color assay Our proposal Send us your pre-initial-therapy sample for assessment of CLL immunophenotype, BCR mutational status and CD38/ZAP70 risk and we send you back the results!
What s missing Cooperation with Czech Repubilc central laboratory!! Quality control management in two centers
How to send your patients sample Collect at least(!) 10 ml heparinzed blood or 5 ml bone marrow aspirate Call TNT pickup Notify central laboratory by sending cover letter via FAX How to pack the sample properly (It s the EU law!) - Demonstration All materials for shipment are provided! 1. Pack tube in tissue paper 2. Put wrapped tube in MedPak pouch bag 3. Seal MedPak 4. Put sample in MedPak and cover letter in transport box Any questions? Contact AGMT
Transport papers
MABTENANCE It s your turn! Contact: g.brachtl@salk.at
Danke schön. III. Medical Department A. Egle, R. Greil Molekularzytologisches Labor L. Haginger AGMT D. Wolkersdorfer, M. Neuwirth Participating centers Dr. Andy Rawstron Leeds, UK Team Molekularzytologisches Labor