Technical requirements for MRD analysis in myeloma: past, present and future

(1)

Technical requirements for MRD

analysis in myeloma:

past, present and future

Andy C. Rawstron

HMDS

St. James’s Institute for Oncology

Leeds Teaching Hospitals NHS Trust

Disclosures:

Honoria/travel support: Celgene, GSK, Roche

Consultancy: Biogen Idec, Gilead

(2)

Response Criteria

• PR:

> 50% reduction of serum M-protein and reduction in 24 hours

urinary M-protein by >90% or to < 200 mg/24 h

– serum/urine M-protein unmeasurable: > 50% decrease in the difference

between involved and uninvolved FLC levels

– Serum/urine M-protein & sFLC are not measurable: > 50% reduction in

plasma cells provided baseline plasma cell percentage was > 30%

– In addition to the above listed criteria, if present at baseline, a > 50%

reduction in the size of soft tissue plasmacytomas is also required

• VGPR:

Serum and urine M-protein detectable by immunofixation but

not on electrophoresis

– or > 90% reduction in serum M-protein plus urine M-protein level < 100

mg/24 h

• CR:

Negative immunofixation on the serum and urine and

disappearance of any soft tissue plasmacytomas and < 5% plasma

cells in bone marrow

• sCR:

CR as defined below plus normal FLC ratio and

absence of clonal cells in bone marrow by immunohistochemistry or

2-4 color flow cytometry

International Myeloma Working Group (IMWG)

Uniform Response Criteria for Multiple Myeloma

(3)

Different technologies for assessing

response and detecting residual disease

• Paraprotein quantitation and immunofixation (PP & IF):

+ Simple serum measurement, widely available, sensitive

– Indirect measurement with variable applicability

– Delayed clearance (IgG half-life ~ 23 days)

• Serum Free Light Chain (sFLC):

– Short half-life



real-time measure of change in tumour burden

– Indirect measurement

– Relatively insensitive (normalises if neoplastic <= normal PC)

• Trephine Biopsy:

– Direct measurement

– Relatively insensitive (1-5%)

(4)

Immunoglobulin Heavy Chain Gene PCR

Transplant

%

amplifiable

LOD

MRDneg /

total

PFS

MRDneg vs.

MRDpos

P

Martinelli

JCO 2000

Allo

88 [0.0001]

17/25

110 vs 35

<0.005

Corradini

Blood 2003

Allo

67 [0.0001]

16/29

NR vs 28

0.0001

Galimberti

Leuk Res 2005

Allo

NS

[0.0001]

8/20

NR vs 8

0.03 Swedin

BJH 1998

Auto

67 [0.0001]

5/10

TREND

0.16 Cremer

BMT 2000

Auto

NS

Relative

13/20

NR vs 19

0.0009

Fenk

Haematologica 2004

Auto

NS

[0.0001]

3/11

NR vs 12

0.01 Bakkus

BJH 2004

Auto

77

0.015 22/60

64 vs 16

0.001 Sarasquete

Haematologica 2005

Auto

75

0.01 7/24

34 vs 15

0.04

(5)

Different technologies for assessing

response and detecting residual disease

• Paraprotein quantitation and immunofixation (PP & IF):

+ Simple serum measurement, widely available, sensitive

– Indirect measurement with variable applicability

– Delayed clearance (IgG half-life ~ 23 days)

• Serum Free Light Chain (sFLC):

– Short half-life



real-time measure of change in tumour burden

– Indirect measurement

– Relatively insensitive (normalises if neoplastic <= normal PC)

• Trephine Biopsy:

– Direct measurement

– Relatively insensitive (1-5%)

• Minimal Residual Disease (MRD)

– Direct sensitive measure of tumour burden

– <0.01% residual disease predicts prolonged progression-free

survival (trend or P<0.05)

(6)

Myeloma MRD: issues with molecular

approaches

• The patient-specific immunoglobulin gene may not be

identifiable with standard approaches

• 

only 75-90% of patients can be monitored

• ASO-RQ IGH-PCR requires patient-specific primers –

i.e. design and validation of the assay for each patient

• Pre-treatment material is required

• 

Difficult to apply the technology to large clinical

trials

• Clinically informative threshold is 0.01%,which is within

the limit of quantitation of flow cytometry

(7)

Myeloma MRD: Flow

• Salamanca:

• 87 patients

• Panel:

CD38/CD56/CD19/CD45

CD138/CD28/CD33/CD38

CD20/CD117/CD138/CD38

• Up to 2 x 10

6 _{cells acquired}

• >30% of plasma cells neoplastic

34 vs. 60 months

San-Miguel (2002)

Blood

99: 1853-6

Induction



ASCT vs. Chemo

(8)

Myeloma MRD: Flow

• UK Myeloma VII

• 45 patients

• Panel:

CD45/CD138/CD38

CD45/CD3/CD38

CD45/CD19/CD38

CD45/CD56/CD38

• Up to 2 x 10

5 _{cells acquired}

• >10% of plasma cells neoplastic

20 vs. 35 months

Rawstron (2002)

Blood

100: 3095-10

CVAMP Induction



HDM ASCT

(9)

Most flow cytometry issues already have a

broad consensus resolution since 2008

www.myeloma-europe.org

• Plasma cell percentage flow vs. morphology and

how to exclude false negative results

• Gating strategy: need for CD38, CD138, and

CD45 in same tube

• Essential, recommended and suggested

markers to discriminate Myeloma from normal

plasma cells: CD19/CD56 + CD117/CD27/CD81

(10)

MRD level by flow cytometry is a better

predictor of outcome than immunofixation

Pavia et al (2008) Blood 112: 4017

GEM2000: VBMCP/VBAD



HDM ASCT

0 25 50 75 100 125 0 20 40 60 80 100

p= 0.002

5-year PFS:

59%

/

49%

/

24%

/

17%

P = 0.002

Months

PFS

MRD

neg

_IF

neg

MRD

pos

_IF

neg

MRD

neg

_IF

pos

MRD

pos

_IF

pos

PFS

MRD

neg

CR MRD

pos

Stringent CR

MRD

pos

PR

0 10 20 30 40 60 0 20 40 60 80 100

p= 0.002

3-year PFS:

90%

/

38%

/

57%

/

28%

P = 0.001

Months

50

Pavia et al (2011)

(11)

Immunofixation is insufficient for multi-stage

treatment strategies incorporating maintenance

96%

68.8%

0

20

40

60

80

100 Remain MRD negative

27.6%

3.4%

0

20

40

60

80

100 Become MRD negative

Thalidomide maintenance

No maintenance

“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).

Morgan et al, Blood 2012, 119(1): 7-15

(12)

Direct quantitative measure of tumour burden

allows better prediction of PFS

(13)

Direct quantitative measure of tumour burden

allows better prediction of overall survival

~1 year improvement in overall survival per log tumour depletion

Myeloma IX 6year survival data – MS in preparation

Median OS:

>1%



4.0yrs

0.1-1%



5.9yrs

0.01-0.1%



6.8yrs

<0.01%



>7.5yrs

(14)

MRD detection in Myeloma: current status

• Serum paraprotein depend on multiple factors (tumour

burden, patient-specific production rate, Ig half-life) - it is an

indirect relative measure with wide variation between patients

• MRD can be readily detected at the 0.01% in a proportion of

patients in CR by IMWG criteria

• Consensus on methodology for flow cytometry and RQ-PCR

- flow cytometry readily applicable to clinical trials

• 3 prospective studies have demonstrated MRD is an

independent predictor of progression-free and overall

survival*

• Flow MRD minimum requirements: CD38/CD138/CD45 with

CD19, CD56, CD117 and CD27 in 1x8-CLR or 2x6-CLR

tubes

* Myeloma IX: PFS published; OS independent when

considered as continuous variable, MS in preparation

(15)

Optimal technique varies according to

the goal of the assessment

Quantitative, direct and sensitive

measure of bone marrow infiltration is

(16)

Improvements in sequencing technology

1970s: The “Gene

Machine”

for amplifying DNA

>500 million

sequencing

reactions on

a chip

Cost in USD ($) per Mb

of DNA Sequence

http://www.genome.g

ov/sequencingcosts/

0.01

0.1

1

10

100 1K

10K

01 02 03 04 05 06 07 08 09 10 11

(17)

Flow Cytometry current standard (4-8 CLR)

Flow Cytometry 2014

consensus (≥8 CLR) RQ-ASO IGH-PCR High Throughput Sequencing

Percentage of patients applicable >95% >95% 70-90% 80-90%

Lower limit of quantification

0.01% 1.0 E-4 1 in 10,000 0.001% 1.0 E-5 1 in 100,000 0.01% 1.0 E-5 1 in 100,000 0.001% 1.0 E-5 1 in 100,000 Lower limit of detection

(sensitivity) 0.004% 4.0 E-5 1 in 25,000 0.0004% 4.0 E-6 1 in 250,000 0.001% 1.0 E-5 1 in 100,000 0.0001% 1.0 E-6 1 in 1,000,000 Approximate number of cells

recommended for LOQ

500,000 events per tube ≥1 million cells

5 million events per tube ≥10 million cells

500ng DNA in triplicate ≥1 million cells

7µg DNA ≥7 million cells Is the assay the same for every

applicable patient?

YES - but additional markers may be required

in up to 10% of cases

YES

NO - requires design and validation of

patient-specific primers

YES

Pre-treatment evaluation Required Preferable Required Required

Does the assay require fresh material

Directly quantitative

NO - patient-specific IGH copy number calibrated to

a standard curve generated from pre-treatment DNA serially

diluted into DNA extracted from pooled donor mononuclear cells

reference IGH sequence, reported as a proportion

of total leucocytes calculated from total DNA

content

Additional check for sample quality

Harmonisation YES (EMN Consensus) Ongoing (ICCS/ESCCA) YES Ongoing (EuroMRD)

Independent prognostic factor for outcome in prospective clinical

trial

No Under evaluation

Progression free and overall survival

PREFERABLE - samples ideally <48hrs old before DNA extraction but analysis can be performed on

archive material; extracted DNA may be stored indefinitely before processing

YES - samples must be <48hrs old and processed immediately

YES - neoplastic plasma cells are reported as a percentage of leucocytes

NOT REQUIRED - identification of hematopoietic elements (progenitors and normal plasma cells)

within the assay

REQUIRED - morphology or cytometry on the sample to assess quality and identify hemodilute

(18)

Patient-specific markers

• Pre-treatment material is generally preferable but not

always available

• Patient-specific assays are not suited to routine

diagnostics

• What is an acceptable proportion of patients suitable for

monitoring

– <75% NO

– >95% YES

– 90% ??

• Martinez-Lopez Blood 2014 ePub

– Sequenta LymphoSIGHT assay: detected a

myeloma-specific gene rearrangement in diagnostic

samples from 91% patients.

(19)

Flow Cytometry 2014

recommended for LOQ

applicable patient?

YES

content

trial

No Under evaluation

within the assay

(20)

Limit of detection / quantification

• 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)

(21)

Limit of detection: small number of target events

leads to >1 log errors on the result

95% CI for a count of

2 events



1.5 log

20 events



0.4 log

50 events



0.25 log

(22)

Quantifying disease levels

• Flow Cytometry:

Number of neoplastic plasma cells as a proportion of

the number of total leucocytes or cells

• RQ ASO-PCR:

Number of cycles to detect amplicon above a certain

threshold calibrated against a standard

• High-throughput sequencing:

– A known quantity of reference IGH sequence is added to the sample and the

number of molecules per sequence read is calculated. The number of

neoplastic sequence read is multiplied by this factor to give the number of

molecules of neoplastic IGH in the original sample

– The total leukocytes in the reaction by measuring the total DNA in the reaction

using standard picogreen methods and RT-

PCR using β actin DNA, assuming

an average human diploid genome mass of 6.49 picograms

– Bioinformatic corrections for – amplification of a given sequence by multiple

primers, and discrimination of replicate amplicons vs. minor clonal expansion,

non-functional rearrangements (generally less than 20% of all VDJ

rearrangements) are included in the analysis

– These metrics were combined to calculate a final MRD measurement, which

is the number of leukemia-derived molecules divided by the total leukocytes in

the sample (capped at 1 million CLL clonotypes per 1 million input PBMC

(23)

High-throughput sequencing: >1 log errors on

MRD count are common

Logan et al.

Leukemia

12 March 2013; doi:

10.1038/leu.2013.52:

ERIC harmonised approach

(24)

When BCR-ABL RQ-PCR accepted as a trial

end-point, 95% LoA = ± 5-fold (0.7 log)

(25)

mpFC and HTS pos

0 1 2 3 4 5 6 7 8 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91 93

mpFC

HTS

mpFC neg, HTS pos

mpFC and HTS neg

1.0 1*10

-1

1*10

-2

1*10

-3

1*10

-4

1*10

-5

1*10

-6

1*10

-7

clonoSEQ Demonstrates Superiority to Flow*

clonoSEQ finds MRD in

32 more patients

Flow missed MRD

at 10

-4

_{in 7 patients}

(26)

Limit of detection: no MRD in a highly sensitive

system is very informative

HTS <

2 neoplastic IGHV sequences in

1 million

total



95% CI for upper limit on count = 6 events



LOD of

0.0006%

(6 x 10-6)

MRD Flow <

20 neoplastic cells in

5 million

total



95% CI for upper limit on count = 30 events

(27)

High-throughput sequencing:

Despite inaccurate quantitation, HTS still highly

predictive of PFS

Logan et al.

Leukemia

12 March 2013; doi:

10.1038/leu.2013.52:

(28)

Flow Cytometry 2014

recommended for LOQ

applicable patient?

YES

content

trial

No Under evaluation

within the assay

(29)

Sample quality: Good vs. Poor

Aspirate/trephine morphology:

Cellularity

Progenitor cell maturation

Aspirate cytometry

Normal CD138+ plasma cells

Normal B-progenitors

Myeloid maturation

Nucleated red cells

RQ-PCR or HTS

No assessment of sample quality

2012 samples at HMDS: 55/1259 (4.3%) follow-up samples

were unsuitable for MRD analysis

(30)

MRD analysis from present to future

• Applicable to a high proportion of patients

– HTS approx 90%, requires pre-treatment material

–

≥6CLR flow cytometry more than 95%

• Sensitive measure of neoplastic cells

– At least 0.01% LoQ for outcome prediction

– 0.001% LoD for current treatment strategies (all technologies)

– <1 in a million preferable (HTS only)

• Quantitative analysis

– All technologies have capacity for discriminating >0.01% vs.

0.001-0.01% vs. <0.001%

– HTS quantification subject to >1 log errors but is the only

technology to detect 1 in a million

• Identifying haemodilute samples to exclude

false-negative results

– ~5% of follow-up samples inadequate for MRD detection

– Flow cytometry incorporates an objective measure of sample

quality

(31)

MRD as a surrogate end-point for clinical trials

• Frontline trials in fit patients



>5 years follow-up

• Direct quantitative measure of tumour burden by flow

cytometry or RQ-PCR (both harmonised) or potentially

high throughput sequencing

• Improved prediction of outcome compared with IMWG

Uniform Response Criteria

– serum markers can be sensitive but at best provide an

indirect

measure of

relative

change

• Prospective studies (n=3) show independent prediction

of PFS and OS using flow cytometry at a 0.01%

threshold

– 0.001% threshold under evaluation but ? a decade to

demonstrate prospectively ?

(32)

University of Birmingham

MT Drayson

K Walker

A Adkins

N Newnham

Wessex Regional Genetics

Laboratory, Salisbury

F Ross

L Chieccio

LTHT, Leeds

G Cook

S Feyler

D Bowen

HMDS, Leeds

RG Owen

AC Rawstron

R de Tute

M Dewar

S Denman

ICR, London

FE Davies

M Jenner

B Walker

D Johnson

D Gonzalez

N Dickens

K Boyd

P Leone

L Brito

A 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

Funding

Medical Research Council

Pharmion

Novartis

Chugai Pharma

Bayer Schering Pharma

OrthoBiotech

Celgene

Kay Kendall Leukaemia Fund

Chief Investigators

JA Child

GJ Morgan

GH Jackson

CTRU, Leeds

K Cocks

W Gregory

A Szubert

S Bell

N Navarro Coy

F Heatley

P Best

J Carder

M Matouk

D Emsell

A Davies

D Phillips

A Gillman

L Flanagan

C Tyas and others