Flow-cytometric quantification of minimal residual disease (MRD) in myeloma: independent outcome prediction & sequential survival benefits per log tumour reduction
St James's Institute of Oncology
Andy C. Rawstron
MRD analysis for clinical trials in myeloma
•Myeloma IX•Using MRD as an endpoint: lessons from the FDA•Harmonisation•Quantitative MRD analysis•Measuring MRD for clinical trials
2
MRC Myeloma IX: Trial design
MRC Myeloma IX: MRD status post-ASCT is an independent predictor of PFS
MRDNEG improved PFS in CR patients (34.3 vs 14.1 months, P=0.0068)
MRDNEG but IFPOS similar to MRDPOS ? Sample quality ? Maintenance randomization
MRDNEG improved OS in CR patients (NR vs 61.9 months, P=0.0928)Best outcome if MRDNEG and IFNEG (P=0.0385)
MRC Myeloma IX: MRD status after induction
Immunofixation response depends on half-life
CVAD CTD
Post induction (n=252) 13% 25% P=0.004
Day 100 (n=397) 54% 71% P<0.0001
LCOM (2-4 hours)
IgA(6 days)
IgG(23 days)
CR post induction
33.3% 20.3% 10.3%
Up to one year to see maximum M-protein responseDavies et al (2001)
Brit J Haem 112:814-9
MRC Myeloma IX: Thalidomide maintenance improves PFS in patients with detectable MRD after HDM
Best outcome was demonstrable in MRD negative patients receiving thalidomide maintenance and worst in those MRD positive patients who did not receive maintenance therapy (P=0.0003)
No change in conventional response with thalidomide maintenance but clear differences in neoplastic plasma cell levels
“Using electrophoresis and immunofixation as a monitoring technique, there was no difference between the thalidomide maintenance and no maintenance arms in the percentage of patients that upgraded response status over time (P .19).” (1)
27.6
96
3.4
68.8
0
20
40
60
80
100
Become MRD negative Remain MRD negative
Thalidomide maintenance
No maintenance(2)
1. Morgan et al, Blood 2012, 119(1): 7-152. Rawstron et al, JCO 2013 in press
Optimal laboratory technique for assessing disease levels varies according to the goal of the assessment
Quantitative, direct and sensitive measure of bone marrow infiltration is optimal for response
assessment
MRD analysis for clinical trials in myeloma
•Myeloma IX• MRD provides rapid and sensitive measure of response to
induction, ASCT and maintenance.
•Using MRD as an endpoint: lessons from the FDA•Harmonisation•Quantitative MRD analysis•Measuring MRD for clinical trials
10
Is MRD suitable as an end-point for clinical trials in myeloma?
•MRD analysis improves assessment of response compared to serum markers alone, particularly in multi-component strategies•Longer survival with increasing treatment options need for biomarkers that predict clinical benefit and offer a rapid measure of treatment efficacy
Flow cytometry detection of minimal residual disease in multiple myeloma: Lessons learned at FDA-NCI roundtable symposiumAm J Hematol. 2014 Dec;89(12):1159-60
Development of “MRD” as a regulatory end-point
• Identify MRD Endpoint in Clinical Trials• 5-10 sub-group analyses, primarily ASO-IGH qPCR
• Develop Assay• Disease-specific flow assay applicable to larger trials
• Standardization of Assay (NIH Consensus Conference)• EMN consensus
• Apply Standardized Assay Prospectively• Apply to Regulatory Action
Study PFS OS Multivariate PFS Multivariate OS
Paiva et al (2008)GEM2000 Yes Yes MRD
Cytogenetics MRD
Rawstron et al (2013)
Myeloma IXYes Yes MRD
Cytogenetics Cytogenetics
Paiva et al (2012)*GEM2000
GEM05<65Yes Yes MRD
CytogeneticsMRD
Cytogenetics
Paiva et al (2011)GEM05>65 Yes No MRD
Myeloma X Yes Insufficient events MRD Insufficient
events
*CR patients only
MRD is an independent prognostic factor for PFS/OS in studies usingCD138 / CD38 / CD45 for gatingCD19 / CD56 / CD27 / CD117 / (CD81) for identifying neoplastic PC
MRD analysis for clinical trials in myeloma
•Myeloma IX• MRD provides rapid and sensitive measure of response to
induction, ASCT and maintenance.
•Using MRD as an endpoint: lessons from the FDA• Would facilitate development of new treatments, needs
harmonisation document and more independent OS data
•Harmonisation•Quantitative MRD analysis•Measuring MRD for clinical trials
14
Harmonised assay for MRD detection
• Characteristics of assays that predict outcome• CD138/CD38/CD45 backbone for gating• CD19/CD56/CD27/CD117/CD81 for characterization
• Reagent specification to permit rapid validation of LDT (lab-developed test) or IVD panels
• Backwards compatible• Suitable for prospective studies
• targeted acquisition of 3-5 million events
MRD analysis for clinical trials in myeloma
•Myeloma IX• MRD provides rapid and sensitive measure of response to
induction, ASCT and maintenance.
•Using MRD as an endpoint: lessons from the FDA• Would facilitate development of new treatments, needs
harmonisation document and more independent OS data
•Harmonisation• Nearly done…
•Quantitative measure of outcome•Measuring MRD for clinical trials
16
Direct quantitative measure of tumour burden allows better prediction of PFS
Direct quantitative measure of tumour burden allows better prediction of overall survival
~1 year improvement in overall survival per log tumour depletionMyeloma IX 6year survival data
Median OS: >1% 4.0yrs 0.1-1% 5.9yrs0.01-0.1% 6.8yrs <0.01% >7.5yrs
Relationship between categorical response and MRD
19
>1% residual disease = PR or worse
20
Direct quantitative measure of tumour burden allows better prediction of outcome for patients in CR
21
1 2 3 4 5 6 7 8
20
40
60
80
100
TIME (YEARS)
% P
RO
GR
ES
SIO
N-F
RE
E S
UR
VIV
AL
<.01% N= 183
.01%-<.1% N= 19
.1%-<1% N= 11>1% N= 1
2
1(TREND) = 10.92
P < .001
1 2 3 4 5 6 7 8
20
40
60
80
100
TIME (YEARS)
% P
RO
GR
ES
SIO
N-F
RE
E S
UR
VIV
AL
1 2 3 4 5 6 7 8
20
40
60
80
100
TIME (YEARS)
% P
RO
GR
ES
SIO
N-F
RE
E S
UR
VIV
AL
<.01% N= 183<.01% N= 183
.01%-<.1% N= 19
.1%-<1% N= 11>1% N= 1
2
1(TREND) = 10.92
P < .001
1 2 3 4 5 6 7 8
20
40
60
80
100
TIME (YEARS)
% O
VE
RA
LL
SU
RV
IVA
L
<.01% N= 183
.01%-<.1% N= 19
.1%-<1% N= 11
>1% N= 1
2
1(TREND) = 4.93
P = .03
1 2 3 4 5 6 7 8
20
40
60
80
100
TIME (YEARS)
% O
VE
RA
LL
SU
RV
IVA
L
1 2 3 4 5 6 7 8
20
40
60
80
100
TIME (YEARS)
% O
VE
RA
LL
SU
RV
IVA
L
<.01% N= 183<.01% N= 183
.01%-<.1% N= 19
.1%-<1% N= 11
>1% N= 1
2
1(TREND) = 4.93
P = .03
Relationship between categorical response and MRD
22
Sample quality
First aspirate morphology Second aspirate laboratory
Differences in aspirate quality according to referring hospital (trial samples)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 (1
0)2
(18)
3 (1
8)4
(13)
5 (2
4)6
(09)
7 (1
5)8
(11)
9 (0
9)10
(09)
11 (1
3)12
(15)
13 (2
7)14
(16)
15 (1
9)16
(25)
17 (1
1)18
(32)
19 (1
4)20
(24)
21 (0
9)22
(09)
23 (0
9)24
(10)
25 (1
0)26
(38)
27 (0
9)28
(24)
29 (1
7)30
(40)
31 (1
9)32
(10)
33 (1
5)34
(37)
35 (2
1)36
(36)
37 (1
9)38
(14)
39 (1
7)
Chart Title
<1% 1-5% 5-10% >10%
Prop
ortio
n of
cas
es
Centre ranking (median % plasma cells from baseline samples)
>10% 5-10% 1-5% <1%Plasma cells
(% of leucocytes)
0%10%20%30%40%50%60%70%80%90%
100%
1 (3
6)
2 (0
8)
3 (1
6)
4 (3
7)
5 (1
5)
6 (2
2)
7 (1
4)
8 (1
0)
9 (3
1)
10 (2
2)
11 (2
3)
12 (2
4)
13 (2
1)
14 (1
3)
15 (4
5)
16 (2
7)
Chart Title
<1% 1-5% 5-10% >10%
Differences in aspirate quality according to referring hospital (diagnostic with trephine)
Prop
ortio
n of
cas
es
Centre ranking (median % plasma cells from baseline samples)
>10% 5-10% 1-5% <1%Plasma cells
(% of leucocytes)
Differences in practice according to the median plasma cell percentage in bone marrow aspirate samples
26
Differences in practice according to the median plasma cell percentage in bone marrow aspirate samples
27
Post-treatment aspirate quality acceptable in >95% of cases, no difference in quality according to baseline sample quality
Prop
ortio
n of
cas
es
<0.01% 0.01-0.1% 0.1-1% 1-10%Neoplastic
plasma cells(% of
leucocytes)
0%10%20%30%40%50%60%70%80%90%
100%
Baseline Median >8%PC (n=40)
Baseline Median <2%PC (n=40)
After Induction
>10%
3 Months after end of treatment
Baseline Median >8%PC (n=26)
Baseline Median >8%PC (n=40)
3 months after ASCT / end of treatment, using % normal PC as a marker of sample quality (>0.01% adequate, >0.1% good):
>8% PC baseline median: 96% adequate / 50% good <2% PC baseline median: 95% adequate / 48% good
Is MRD relevant in PR?
Quantitative MRD and cytogenetics are independent predictors of progression-free and overall survival
30
Outcome depends on disease level not treatment
Outcome depends on disease level not treatment
32
CVAD (n=91)
CVAD (n=117)
CTD (n=123)
CTD (n=66)
Proportion of patients
0%
20%
40%
60%
80%
100%
CR Not CR
Number of patients
020406080
100120140
CVAD (n=91)
CVAD (n=117)
CTD (n=123)
CTD (n=66)
CR Not CR
<0.01% 0.01-0.1% 0.1-1% 1-10%Neoplastic
plasma cells(% of
leucocytes)
>10%
Achieving <0.01% MRD: impact of ASCT
•Median (range) % neoplastic plasma cells at end of induction, data available in 253/397 cases, of which 47/253 had <0.01% MRD after induction and ASCT
• ≥0.01% MRD after ASCT (n=96) 1.5% (0.02 – 25%)
• <0.01% MRD after ASCT (n=110) 0.02% (0.02 – 14%)
•≥0.01% MRD after induction & ASCT Median 0.67 log tumour depletion (range -1.4 – 2.6)
•≥0.01% MRD after induction & <0.01% MRD after ASCT Median >1.7 log tumour depletion
•Responsive patients achieve ~2log depletion to ASCT
•>1% MRD after induction unlikely to respond optimally
33
MRD analysis for clinical trials in myeloma
•Myeloma IX• MRD provides rapid and sensitive measure of response to
induction, ASCT and maintenance.
•Using MRD as an endpoint: lessons from the FDA• Would facilitate development of new treatments, needs
harmonisation document and more independent OS data
•Harmonisation• Nearly done…
•Quantitative measure of outcome• independent predictors of progression-free and overall survival• Sample quality acceptable – first (or only) pull for lab studies
•Measuring MRD for clinical trials
34
High-throughput sequencing: >1 log errors
Logan et al. Leukemia 12 March 2013; doi: 10.1038/leu.2013.52:
ERIC harmonised approach
Leukemia 2007, 21(5): & 2013, 27(1)
MRD by high-throughput sequencing
• Isolate DNA and combine with 3 IGHV reference standards• 1º PCR: 16 cycle amplification of IGHV with consensus V and J primers (optimised to minimally skew the repertoire frequency during amplification) and append annealing sites for 2º PCR primers
• Second stage PCR: 22 cycles using 1/100 of the 1º PCR product append sample indices and cluster formation sequences
• Pool samples and purify (QIAquick)• Amplify in situ on Illumina via bridging PCR and sequence• MAP sequences to IMGT database and correct for
• differential amplification of IGHV rearrangements• replicate amplicons and minor clonal expansions• non-functional rearrangements
• Calculate the number of neoplastic (and total B-cell) reads using the IGHV reference standard
• Calculate total leukocytes (total DNA by picogreen and qPCR for β-actin)
High throughput sequencing for MRD detection: negative result substantial improvement in outcome
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4023416/
MRD strategy for UK clinical trials
•Median 30 million cells per BM aspirate•Flow 10-4 (LoD 0.002%) 2 million cells
• Suitable LoD for substantial proportion of cases• CD138/CD38/CD45 +
CD19/CD56/CD27/CD117/CD81
•DNA for HTS 10 million cells•Immunomagnetic CD138-selection and storage of CD138+ and CD138- fractions for HTS 10 million
•If required and sufficient cells, flow 10-5 10 million
MRD analysis for clinical trials in myeloma
•Myeloma IX• MRD provides rapid and sensitive measure of response to
induction, ASCT and maintenance.
•Using MRD as an endpoint: lessons from the FDA• Would facilitate development of new treatments, needs
harmonisation document and more independent OS data
•Harmonisation• Nearly done…
•Quantitative MRD analysis• independent prediction of progression-free and overall survival• Sample quality acceptable – first (or only) pull for lab studies
•Measuring MRD for clinical trials• Combination of flow + HTS MRD optimal
39
University of BirminghamMT DraysonK WalkerA AdkinsN Newnham
Wessex Regional Genetics Laboratory, SalisburyF RossL Chieccio
LTHT, LeedsG CookS FeylerD Bowen
HMDS, LeedsRG OwenAC RawstronR de TuteM DewarS Denman
ICR, LondonFE DaviesM JennerB WalkerD JohnsonD GonzalezN DickensK BoydP LeoneL BritoA Avridromou
MRC Leukaemia Trial Steering Committee
MRC Leukaemia Data Monitoring and Ethics Committee
NCRI Haematological Oncology Clinical Studies Group
NIHR, through the National Cancer Research Network
UK Myeloma Forum Clinical Trials Committee
Myeloma UK
FundingMedical Research CouncilPharmion Novartis Chugai Pharma Bayer Schering PharmaOrthoBiotech CelgeneKay Kendall Leukaemia Fund
Chief InvestigatorsJA ChildGJ MorganGH Jackson
CTRU, LeedsK CocksW GregoryA SzubertS BellN Navarro Coy
Acknowledgements
41 Thanks!