Kerrie Clerici, Michael Swain, Dominic Fernandez, Julia Schulz, Matthew Archer, Janine Campbell

Transcription

1 Minimal Residual Disease (MRD) Testing by Flow Cytometry for childhood Precursor B Cell Acute Lymphoblastic Leukaemia Royal Children s Hospital experience. Kerrie Clerici, Michael Swain, Dominic Fernandez, Julia Schulz, Matthew Archer, Janine Campbell

2 Children s Oncology Group The Children s Oncology Group (COG) studied the prognostic impact of MRD at different time points in a large cohort of patients. Samples at 3 different time points were assessed. Peripheral blood at Day 8 of induction Bone Marrow Aspirate at Day 29 of induction Bone Marrow Aspirate at end of consolidation Borowitz M J et al. Blood 2008; 111(12):

3 Day 8 Day 29 Borowitz M J et al. Blood 2008; 111(12):

4 Prognostic significance of day 8 blood MRD in patients who are MRD negative in bone marrow by day 29 Borowitz M J et al. Blood 2008; 111(12):

5 Prognostic significance of endconsolidation MRD Borowitz M J et al. Blood 2008; 111(12):

6 Multivariate analysis of prognostic factors affecting outcome Variable Hazard ratio P Day 29 MRD>0.01% 4.31 <0.001 NCI risk group 2.25 <0.001 Trisomies 4 and <0.001 Day 8 MRD (PB) >0.01% TEL-AML Day 8 M1 marrow Borowitz M J et al. Blood 2008; 111(12):

7 Overview of the COG AALL08B1 Classification System for B-precursor ALL Risk Group Low Average High Very High Projected 5-yr EFS > 95% 90-95% 75-90% < 75% NCI Risk-Group SR SR SR SR SR HR SR HR Any Favorable Genetics Yes Yes No Yes No Any No Any No Day 8 PB MRD < 0.01% 0.01% < 1.0% Any 1.0% Any Any Any Any Day 29 BM MRD < 0.01% < 0.01% < 0.01% 0.01% < 0.01% < 0.01% >0.01% >0.01% Any Patient number/ year Fraction of patients 14.7% 37.3% 35% 13% Source: Children s Oncology Group, AALL08B1. Classification of Newly Diagnosed Acute Lymphoblastic Leukaemia (ALL). Version 05/28/10

8 Our experience Received a Government New Technology grant in Sept 2010 Visited the COG Flow Cytometry Reference Laboratory in Seattle, Nov 2010 and again in May 2013 Navios, 10 colour flow cytometer arrived in Dec 2010 And so the fun began... We tested our first patient in March 2011 (test in validation phase) We reported our first result in Sept We are now an approved COG reference centre Sept 2015

9 Method Based on COG procedure Stain-then-lyse/fix procedure 1 x 10 6 cells labelled per tube Samples were run on a Navios TM Flow Cytometer (Beckman Coulter) and analysed using Kaluza (Beckman Coulter) software Aimed to collect at least MNC events ( total events) so we can achieve a sensitivity of at least 0.01% Flow Set Pro beads (Beckman Coulter) were used to standardise fluorescent parameters Titrations performed on all antibodies and then each new lot was verified to ensure reproducible fluorescence and optimal staining and compensation check.

10 Panel Fluorochrome Tube 1 Tube 2 Tube 3 (Denominator) Denominator calculation: MRD (T1 or T2) / B cells (T3) (for BM samples) B cells (T1 or T2) / mononuclear cells (T3) Denominator for PB (day 8 samples) is Syto 16 (TNC) Day 8 Tube (PB) FITC / Syto 16 CD20 CD9 Syto 16 Syto 16 PE CD10 CD13 & CD33 CD20 PerCP-Cy5.5 CD38 CD34 CD3 CD34 PC7 CD19 CD19 CD19 CD19 APC CD58 CD10 CD71 CD10 APC-H7 CD45 CD45 CD45 CD45

11 Denominator Use a denominator that is meaningful to your institution and / or the treatment protocol. Denominators = 1. Total White Blood Cells - CD45 pos 2. Total Nucleated Cells (TNC) - DNA stain, includes NRBC 3. Mononuclear cells (MNC) - DNA stain, includes NRBC 0.019% of CD % of TNC 0.011% of MNC

12 Denominator High regenerating myeloid cells affecting MRD % 0.40% of CD % of TNC 1.99% of MNC Molecular MRD = 0.6% (frozen) High NRBC affecting MRD % 0.05% of CD % of MNC 0.009% of TNC

13 Normal Template: Tube 1

14 Normal Template: Tube 2

15 Results n = 261 patients (15 adults), >750 MRD tests Day 8: 84% MRD positive (> 0.01%), 27% at a level >1% Day 29: 30% MRD positive (> 0.01%) This agrees well with the reported level of positivity in the literature.

16 Results Most positive cases showed a discrete cluster of at least 20 abnormal cells. 245/261 (94%) patients had aberrant phenotypes that could be distinguished from normal haematogones 9 patients are now being monitored post bone marrow transplant. 1 patient is enrolled on a clinical trial involving CAR T cells due to a positive MRD at day 8, 29 and end of consolidation therapy combined with unfavourable cytogenetics.

17 patient Day 8 MRD% Day 29 MRD % End consolidation MRD % Results Transplant patients Reason for Transplant poor cyto(mll t(4;11), MRD+, matched sib 2 NT NT NT Relapse post 1 st BMT (6.5& 20.8) (0.09) 3.99 (20& 55.9) NT HR, poor cyto (BCR-ABL) Relapse, matched sib Pre BMT MRD % Day 60 MRD% Day 100 MRD % (pos <0.005) 0.01 (0.0009) HR, Relapse HR, poor cyto (near haploid) matched sib (75.1) (35 &22.5) HR, poor cyto (BCR-ABL), poor response 8 NT NT NT Relapse, matched rel CSF relapse, poor cyto (BCR-ABL) 0.32 (2 & 1.1) (pos <0.001 <0.16 (insuff.) (pos<0.005 & 0.00) (pos <0.005) pending (Neg& 0.0%) (pos ) Day 200 MRD % 1 year MRD % NT 0.04 (0.02) (Neg& 0.0%) D132 Relapsed pending pending

18 Correlations Flow Cytometry N=125 COG enrolled patients (direct comparison with COG flow cytometry data) N=119 Day 8 data pairs 109 in agreement (pos or neg at 0.01% cut off, or clinically significant cut off of >1%) 10 (7 reviewed to agree) discrepant (RCH >0.01%, COG % or RCH>1%, COG <1%) 109/119, 92% concordance N=118 Day 29 data pairs 109 in agreement (pos or neg at 0.01% cut off) 9 (9 reviewed to agree) discrepant (RCH >0.01%, COG %) 109/118, 92% concordance 9 patients have changed risk groups and treatment based on the RCH result.

19 Day 8 Correlations with COG Flow Cytometry Results % of abnormal cells detected by RCH R² = % of abnormal cells detected by COG % of abnormal cells detected by RCH Removing 3 outliers 3 outliers (>20% (>20% MRD) MRD) % of abnormal cells detected by COG

20 Day 29 Correlations with COG Flow Cytometry Results % of abnormal cells detected by RCH R² = % of abnormal cells detected by COG % of abnormal cells detected by RCH Removing 2 outliers Removing (>0.25%) 4 outliers (>1% MRD) % of abnormal cells detected by COG

21 Correlations Molecular N=101 Day 29 and post consolidation comparison with molecular results 98 in agreement (pos or neg at 0.01% cut off) 3 discrepant (1 flow cytometry neg, molecular pos) 98/101, 97% concordance 2 flow cytometry pos, molecular neg

22 Correlations with Molecular Results % of abnormal cells detected by molecular R² = % of abnormal cells detected by Molecular Removing 4 high % MRD outliers % of abnormal cells detected by flow cytometry % of abnormal cells detected by flow cytometry

23 Conclusion What lessons have we learnt? It takes time to become experienced It is worth taking the time to set up a robust panel and maintaining a high level of quality control. Robust, rapid, cost effective We are giving all our patients the benefit of MRD based risk stratification of their treatment. Enables accurate monitoring post transplant to tailor immunosuppressive therapy. Challenges Anti-CD19 therapies

24 Acknowledgements Cell Therapy and Flow Cytometry Laboratory Michael Swain Domenic Fernandez Julia Schulz Matthew Archer Haematologists Janine Campbell Helen Savoia Children s Cancer Centre, RCH Francoise Mechinaud COG Data manager, RCH Allison Lamb Angie Cutajar Brenton Ward Lyn Verkerk COG reference laboratory Brent Wood Katy Dough