Flow-cytometric quantification of minimal residual disease (MRD) in myeloma: independent outcome prediction & sequential survival benefits per log tumour

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


•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




•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






•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



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







•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







•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!

Documents

Flow-cytometric quantification of minimal residual disease (MRD) in myeloma: independent outcome prediction & sequential survival benefits per log tumour