mrd in cll - ericll.org · fewer than one cll cell per 10,000 leukocytes •the blood can generally...

MRD in CLL Andy C. Rawstron

HMDS

Leeds Teaching Hospitals NHS Trust

Improving Outcomes in CLL

Adapted from Montserrat et al. Blood 2005;106:2226

% O

bje

cti

ve R

esp

on

se

0

20

40

60

80

100

1970 1980 1990 2000 2010

Ch

lora

mb

uc

il

COP

CHOP

Fludara

Fludara

combo

FCR

Impro

ved

OS

Impro

ved

PF

S

Sym

tpom

palli

ation

0 2 4 6 8 10

0.0

0.2

0.4

0.6

1.0

0.8

Stage B/C

1980-1994

1995-2004

Year of Diagnosis Rela

tive S

urv

ival

Years from Diagnosis

The value of response assessment in CLL

Hallek et al Lancet 2010; 376: 1164–74

• Currently high CR rate (>70%)

and long survival (>5yrs)

• Many new agents but the

pace of demonstrating clinical

benefit has slowed

Figure 2 Progression-free survival in all patients (A) and in patients with Binet stage B and C chronic lymphocytic leukaemia (B)

Chemoimmunotherapy=fludarabine, cyclophosphamide, and rituximab. Chemotherapy=fludarabine and cyclophosphamide.

M Hallek , K Fischer , G Fingerle-Rowson , AM Fink , R Busch , J Mayer , M Hensel , G Hopfinger , G Hess , U v...

Addition of rituximab to fludarabine and cyclophosphamide in patients with chronic lymphocytic leukaemia: a randomised, open-label,

phase 3 trial

The Lancet Volume 376, Issue 9747 2010 1164 - 1174

http://dx.doi.org/10.1016/S0140-6736(10)61381-5

Progression-free survival in DCLLSG CLL 8 trial

CLL trials assessing MRD as a predictor of survival

Robak et al. [8] Cladribine + C Untreated 82 88 24 75 CD19/CD5 NA

Robertson et al. [4] FP Unselected 159 60 12 81 CD19/CD5 >30 vs 19‡

Keating et al. [2] FCR Untreated 224 95 70 78 CD19/CD5 >48 vs 39

Montillo et al. [9] F/F→A Untreated 34 100 79 56 IgH PCR NA

Del Poeta et al. [10] F→R Untreated 60 93 78 93 3-color flow >30 vs 24

Wendtner et al. [11] F/FC→A Untreated 21 100 27 83 ASO-PCR >25 vs 25§

Bosch et al. [5] FCM Relapsed/refractory 60 78 50 34 4-color flow 21 vs 17

Moreton et al. [6•] A¶ Relapsed/refractory 91 54 35 57 4-color flow >36 vs 20

O’Brien et al. [12] Various→A Relapsed/refractory 41 46 36 38 ASO-PCR >24 vs 15

Elter et al. [13] FA Relapsed/refractory 36 83 31 47 ASO-PCR NA

Wierda et al. [3] FCR Relapsed/refractory 177 73 25 32 ASO-PCR 44 vs 27

Milligan et al. [14] F**→auto Untreated 115 65 42 80 IgH PCR >33 vs 38

Moreno et al. [15••,16] Auto Relapsed/refractory 27 100 85 68 IgH PCR 73 vs 16

Moreno et al. [15••,16] Auto Relapsed/refractory 27 100 85 59 4-color flow 75 vs 16

Ritgen et al. [17] Auto Relapsed/refractory 26 NA NA 23 ASO-PCR Not stated

Moreno et al. [15••,16] Auto Relapsed/refractory 27 100 85 29 ASO-PCR >60 vs 19

Sorror et al. [18] Allo Relapsed/refractory 64 56 13 0, 100 IgH PCR NA

Caballero et al. [19] Allo Relapsed/refractory 30 70 87 68, 94 4-color flow NA

Ritgen et al. [17] Allo Relapsed/refractory 9 NA NA 0, 78 ASO-PCR NA

Moreno et al. [15••,16] Allo Relapsed/refractory 23 91 91 23, 61 ASO-PCR NA

MRD analysis improves prediction of progression-free survival

• >20 trials with some form of MRD analysis

• All show improved PFS for MRDNEG response

• Approximately 1-2 years improvement in PFS for CRNEG vs CRPOS

• Wide variety of assays (consensus PCR, ASO-PCR, flow

clonality, CD19/CD5, disease-specific MRD flow

Percent in

remission at 2

years

0

20

40

60

80

100

PR CR: MRDPOS CR: MRDNEG

DCLLSG CLL FC vs. FCR: MRD level predicts outcome independent of treatment arm

Böttcher, JCO 2012

• Both 4-colour flow cytometry (MRD flow) and allele-specific oligonucleotide PCR are reliably sensitive down to a level of approximately one CLL cell in 10,000 leukocytes

• As such, patients will be defined as having a clinical remission in the absence of MRD when they have blood or marrow with

fewer than one CLL cell per 10,000 leukocytes

• The blood can generally be used to make this assessment except within 3 months of completing therapy, particularly for patients treated with alemtuzumab, rituximab, and other antibodies targeting CLL

Minimal Residual Disease Threshold in CLL – iwCLL Guidelines 2008

Hallek M, et al. Blood 2008; 111:5446–5456. iwCLL, International Workshop on CLL; PCR, polymerase chain reaction.

Assuming Exponential Growth at the MRD Level Linear Increase in PFS per Log Tumour Depletion

CR, complete remission; PR, partial remission.

Ab

solu

te N

um

be

r o

f C

LL C

ell

s (1

09/L

)

Years from End of Therapy

0.01

0.1

1

10

0.001

0.0001

2 3 4 5 0 1 6 7

CLL doubling

time

Extra PFS per

log depletion

6 months 1.7 yrs

3 months 0.8 yrs

1 month 0.3 yrs

PR

CR

Kinetics of Relapse: Exponential Growth from the Lowest Detectable MRD Level

2

Time (Years)

4 6 8 10

Serial MRD measurements in a cohort of 32 MRD+ patients in clinical remission with no absolute

lymphocytosis after treatment [predominantly FCR] at Leeds

Total 68 patients monitored, 31 persistent MRD <0.01%, 5 insufficient MRD+ timepoints.

0.01

0.1

1

10

100

0.001

0.0001

Ab

solu

te N

um

be

r o

f

CLL

Ce

lls

(10

9/L

)

B cell count 5 x 109/L

ADMIRE/ARCTIC Trial (FCR-Based Treatment): Sequential Benefit in PFS per Log Reduction in MRD

Rawstron AC, et al. XVI iwCLL Annual Meeting 2015. FCR, fludarabine, cyclophosphamide, rituximab.

33% (95% CI = 27–38) risk reduction for disease progression per log reduction in MRD level

Progression-free Survival by bone marrow MRD level at 3 months post treatment

N=343 × × × × × × ×

× ×

× × ×

× × ×

× × × ×

× × ×

× × ×

× × × ×

× ×

×

× × ×

× × × × ×

× × × × × × × × × × × ×

× × × × × × × × × × × × × × × × × × × × × × ×

× ×

× × × × × × × × × × × ×

× × ×

×

× × × ×

×

×

×

×

× × × ×

×

×

×

<0.01%

<0.1%

<1%

<10% ≥10%

Log-Rank 2

4 = 224.125 p<0.0001

0 6 12 18 24 30 36 42 48 54 60

1.0

0.8

0.6

0.4

0.2

0.0 Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

MRD in CLL: use as an endpoint in clinical trials

• There are a number of new approaches to treatment but determining the optimal approach will take many years for randomised controlled trials to evaluate progression-free survival (PFS) or overall survival (OS) and using MRD (at 3 months post-treatment) as a trial endpoint could greatly speed the process.

Public Workshop on Minimal Residual Disease

(MRD) as a Surrogate Endpoint in Chronic

Lymphocytic Leukemia (CLL)

www.fda.gov/Drugs/NewsEvents/ucm340707.htm

www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/12/WC500179047.pdf

MRD Analysis Requires a Quantitative Method That Is Not Influenced by the Polyclonal Background

with Prospective Validation

• Flow cytometry: Multiparameter assessment of CLL phenotype that is not clonality based

• PCR: Real-time quantitative ASO-PCR using patient-specific primers, not consensus primers

• High-throughput sequencing: Amplification of all B-cell sequences and enumeration of CLL-specific immunoglobulin gene

Development of M‘D as a

regulatory endpoint:

Identify MRD endpoint in clinical

trials

Develop assay

Standardisation of assay

Apply standardised assay

prospectively

Apply to regulatory action

1995

2007

2012

2002

Guidelines on the validation of cell-based assays

• Sensitivity

• Eithe : lo est sig al dete ta le a o e a kg ou d

• O : t ue positi e / t ue positi e + false egati e

• 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 Quantitation (LoQ) = lowest level of measurand that can be reliably detected and whose total error (bias + Imprecision) meets a desired criterion for accuracy (clinical utility)

Clinical and Laboratory Standards Institute

(CLSI) has published the guideline EP17,

Protocols for Determination of Limits of

Detection and Limits of Quantitation.

UKAS ISO15189 guidelines

Clinically appropriate level of variation: When BCR-ABL RQ-PCR accepted as a trial end-point,

95% limit of agreement = ± 5-fold

CML: best 95% LOA +/- 2-fold using synthetic standards

CLL: Target: ±3-fold (0.5log), preferably ±2-fold (0.3 log)

Flow Cytometry MRD Detection

Rawstron AC, et al. Leukemia 2016; 30:929-936; Rawstron AC, et al. Leukemia 2013; 27:142–149; Rawstron AC, et al. Leukemia 2007; 21:956–964.

Parameters Tubes Detection

limit

Cells required for

0.01% – LoD

6 (4-colour) 4 0.005% 4–20 million

8 (6-colour) 2 0.001% 2–10 million

10 (8-colour) 1 0.001% 1–5 million

V450 V500 FITC PE PerCP-Cy5.5

PE-Cy7

APC APC-

H7

CD

5

CD

3

CD

81

CD

79b

CD

22

CD

19

CD

43

CD

20

Mary Sartor Sydney 10-colour flow assay

Much better reagents became available

Requests to include: CD45, CD38, CD160,

CD200, CD305, ROR1 LoD, limit of detection. .

Rawstron AC, et al. Leukemia 2016; 30:929–936.

Antigen Typical

expression

(% positive

vs control)

Control population in

normal peripheral blood

Minimum relative

fluorescence

intensity

(preferred) Positive Negative

CD5 Positive (>20%) CD3+

T-cells

CD19+

B-cells

>30 (>65)

CD20 Weak CD19+

B-cells

CD3+

T-cells

>10 (>20)

CD43 Positive (>20%) CD3+

T-cells

CD20+

B-cells

>15 (>40)

CD79b Weak CD20+

B-cells

CD3+

T-cells

>15 (>30)

CD81 Weak CD3+

T-cells

Granulocytes >12 (>20)

Weak indicates % reduction in fluorescence intensity relative to the median expression

observed with a reference population of polyclonal B-cells using the same antibody. The

minimum relative fluorescence intensity would provide separation of CLL cells from

normal B-cells in >95% of cases with a preferred relative fluorescence intensity being the

level at which 99% of cases have optimal separation of CLL cells from normal B-cells.

‘e ui es ≥6 a ke s to achieve 0.01% –

available to most labs

The core panel must meet these 6 specifications, but

is flexible thereafter

Rawstron AC, et al. Leukemia 2016; 30:929–936 (Suppl). PB, peripheral blood.

* Following treatment cessation.

PB

PB*

BM

Examples of MRD analysis in patients

treated with non-FCR regimens ‘e ui es ≥6 a ke s to achieve 0.01% –




Backwards-compatible and applicable to current

treatments

Progression-Free Survival by Bone Marrow MRD Status and iwCLL Response with FCR-Type Therapy

Rawstron AC, et al Haematolgica 2015; 100 (S1):Abstract S794.

‘e ui es ≥6 a ke s to achieve 0.01% –




Backwards-compatible and applicable to current

treatments

Validated prospectively in FCR-based treatment 9 MRD-negative PR: 5 splenomegaly, 3 nodes >1.5 cm,

none with BM or >1 site involved

Months from Randomisation

Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

0 12 6 42 48 60 54 18 24 36 30

0.8

0.6

0.4

0.2

0.0

1.0

0.7

0.5

0.3

0.1

0.9

Log-Rank

χ23 = 51.7696

p<0.0001

1: MRD+ CR

2: MRD+ PR

3: MRD- CR

4: MRD- PR

ADMIRE and ARCTIC

BM MRD (N=329)

Flow Cytometry and qPCR


ASO, allele-specific oligonucleotide; IgH, immunoglobulin heavy; qPCR, quantitative polymerase chain reaction; RQ, real-time, quantitative.

• Patient-specific ASO IgH gene PCR

• (Quasi) Quantitative analysis

• Calibrate to standard curve generated by serial dilution of pre-treatment material into normal DNA

• Previously more sensitive than flow cytometry (10-5–10-6); good correlation to 10-4

• The assay has to be validated for each patient (not because of the primer design requirement)

ERIC harmonised approach

<0.001 0.001 0.01 0.1 1 10 100

100

10

1

0.1

0.01

0.001

<0.001

CLL Cell Percentage: MRD Flow Cytometry

CLL

Ce

ll P

erc

en

tag

e:

RQ

-AS

O I

gH

-PC

R

• The sensitivity of an RQ-PCR assay is dependent on several factors, including the type of rearrangement, the size of the junctional region and the amount of DNA in each reaction

• standard curve, including the lowest dilutio of the ua titati e a ge and all previous dilutions:

• must include at least three dilution points

• must have a minimum range of two logs

• must have a slope between -3.1 and -3.9; and

• must have a correlation coefficient . .

• sdfsd

Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-

time quantitative PCR data

Vincent van der Velden et al, Leukemia (2007) 21, 604-611


• The sensitivity is the lowest dilution that meets all the following criteria:

• specific amplification, as determined by the shape of the amplification curve

• o e positive replicate

• lowest CT alue 1.0 lower than the lowest CT value of the background

• lowest CT value <20 cycles from the undiluted sample or, if this undiluted sample is not included in the standard curve, from the intercept of the standard curve (representing the 100 dilution).




Sensitivity Limit of detection


• Quantitative range

• Specific amplification (shape of amplification curve)

• Reproducible amplification (replicate ΔCT <1.5)

• All CT values >3.0 lower than the lowest CT value of the background

• Mean CT value must be within a defined range from the mean CT value of the previous dilution point:

• 2.6–4.0 CT between 10-fold dilutions (e.g. 10-3 to 10-4)

• 0.5–1.5 CT between two-fold dilutions (e.g. 10-3 to 5x10-4)


Quantitative range Limit of quantitation



• RQ-PCR data should be corrected for the a ou t a d amplifiability of the DNA of the diagnosis and the follow-up samples by analyzing a control gene in parallel to the Ig or TCR gene target.

• Quantitation is performed:

• using the standard curve, excluding dilutions outside the ua titati e a ge , of the i ol ed Ig/TC‘ ta get;

• using the mean CT of the triplicates of the follow-up sample and

• correcting the MRD level according to the DNA quality/quantity of the diagnostic sample and the follow-up sample as determined by RQ-PCR of the control gene.

• asdf




High-Throughput Sequencing with Universal Reagents (IGH-VDJ, IGH-DJ, IGK) Acceptable Linearity to 1 in 10-6


Case 1

Case 2

Case 3

Sequence 1 (productive)

Sequence 2 (non-productive)

Average of sequences 1 and 2

10

1

0.1

0.01

0.001

0.0001

0.00001

0.00001 0.0001 0.001 0.01 0.1 1 10

CLL

% L

eu

kocy

tes

Usi

ng

Ad

ap

tiv

e C

lon

oS

EQ

Expected CLL % Leukocytes

HTS: likely to predict very good outcome for MRD <10-6, reproducible but can have >1log difference from RQ-PCR

Logan AC, et al. Leukemia 2013; 27:1659–1665.

100 101 102 103 104 105 106 0 IGH-HTS CLL Cells per 106 Genomes

AS

O-P

CR

CLL

Ce

lls

pe

r 1

06 G

en

om

es

100

101

102

104

106

0

105

103

r=0.64

p<0.0001

12 36 48 60 72 84 96 0 24 Months after Transplant

Dis

ea

se-F

ree

Su

rviv

al

(%)

20

40

60

80

100

0 p=0.0002

MRD <10-6

M‘D ≥ -6

CAP accredited & CLIA approved

Issues to be resolved for quantitative HTS using universal primers

Correction factors

• Calculations based on addition of reference DNA

• Estimation of total leucocytes,

• Amplification bias probably requires calculation of quantitative range per patient as RQ-PCR

• Replicate amplicons and non-functional rearrangements

False positive results

• ERIC: no CLL-asso iated se ue es present in an unrelated sample at the level of0.010% or greater,

• Four CLL-associated sequences were detected in one or more unrelated cases, representing a median 0.00080% (range 0.00046-0.0019%)

Prospective Validation Using synthetic templates to design an

unbiased multiplex PCR assay. Nat Commun.

2013;4:2680.

Selection of techniques for measuring MRD as a clinical trial endpoint


• Requires a quantitative method that is not influenced by the

polyclonal B-cell background

• PFS/OS benefit is detectable with a 1-2 log depletion

MRD assay is ideally quantitative to ±0.3 log

• Prospective validation

• multi-parameter cytometry and RQ-PCR

• Validated assays e ui e li it of dete tio for each result

stated on the report

• Cellular assays: number of events acquired

• Molecular assays: total DNA, patient-specific IGH effects

• Co i atio of oth 6-CLR flow cytometry and RQ-PCR/HTS

BCR-pathway inhibition: different pattern of disease depletion

MRD suitable as an endpoint for therapies aimed at

disease eradication, not disease control:

evaluate at response assessment after treatment

0

20

40

60

80

100

120

140

160

Dec-12 Jun-13 Jan-14 Jul-14 Feb-15 Aug-15 Mar-16

WBC

Monitoring CML response to Imatinib: the white cell count is not informative

Normal CBC MMR

Monitoring CML response to Imatinib: inhibitor dependent on molecular response

Imatinib Nilotinib

CLL: Identifying combinations which achieve <0.01%

MRD in the majority of patients

Measuring the kinetics of response to BCR-pathway inhibitors

0.001

0.01

0.1

1

10

100

1000

0 6 12 18

0.001

0.01

0.1

1

10

100

1000

1 2 6 9 12 18

Ab

solu

te C

LL C

ell

Co

un

t

(10

9/L

)

Months of Ibrutinib Treatment

IcICLLe: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB

Measuring the kinetics of response to BCR-pathway inhibitors

0.001

0.01

0.1

1

10

100

1000

0 6 12 18

Ab

solu

te C

LL C

ell

Co

un

t

(10

9/L

)

Months of Ibrutinib Treatment

IcICLLe: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB

Optimal response

assessment requires

MRD analysis (or at

least high-sensitivity

B-cell enumeration)

?? Similar to CML ??

• GALACTIC: GA101 (obinutuzumab) monocLonal Antibody as Consolidation Therapy In CLL

• Phase 2/3, multicentre, RCT testing obinutuzumab versus no consolidation in CLL patients with >0.01% MRD at 3–12 months after 1st–3rd-line therapy

• Stopping treatment: 6 months MRD <0.01% in PB (3 assessments), confirmed in BM end treatment

• FLAIR: Ibrutinib + rituximab versus FCR (n=754)

• IcICLLe: Ibrutinib monotherapy (n=40)

• CALiBRe: Idelalisib monotherapy (n=40)

• IcICLLe extension: Ibrutinib + obinutuzumab (n=40)

• CLARITY: Ibrutinib + venetoclax (n=50)

Guiding Treatment

GALACTIC https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-000880-42/GB (accessed May 2016); FLAIR: https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-001944-76/GB (accessed May 2016);

IcICLLe & extension: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB (accessed May 2016); CLARITY: http://www.isrctn.com/ISRCTN13751862 (accessed May 2016).

Compartment effect

Rawstron AC, et al. Haematolgica 2015; 100(S1):Abstract S794 (oral presentation).

• Discrepant peripheral blood and bone marrow in all chemoimmunotherapy trials

• Discrepancy frequency varies from 7% to 57% of samples per trial

Trial Treatment n MRD

assesment

Paired PB &

BM samples

(n)

N° with MRD

BM >0.01%

& PB <0.01%

(%)

N° with

>2 log

difference

between PB

& BM (%)

Median log

difference*

(range)

CLL 202

CAMFlud, CLL

207

Alemtuzumab

+/-

Fludarabine

97 End of

treatment 95 16 (16.8%) 5 (5.2%)

1.7

(-1.3 to 3.3)

CLL 201,

MO20927,

ADMIRE +

ARCTIC

Rituximab +

FC or FCM 567

3M post end

of treatment 333 68 (20.4%) 3 (0.9%)

0.69

(-1.3 to 2.4)

CALiBRe, IcICLLe Idelalisib,

Ibrutinib 60 1M & 6M 112 0 (0%) -

0.0

(-0.72 to 1.1)

The Outcome for Patients with MRD Detectable in BM but Not in PB (PB - / BM +) Differs According to

Type of Therapy


PFS for PB-BM+

similar to PB+BM+

Alemtuzumab therapy

PFS for PB-BM+ PFS similar to

PB-BM- in the short term


Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

0.2

0.4

0.6

0.8

1.0

0.0

6 12 18 24 30 36 60 0 42 48 54

Log-Rank

22 = 112.565

1: BM + / PB +

2: BM + / PB -

3: BM - / PB -


Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

6 12 18 24 30 36 60 0 42 48 54

0.2

0.4

0.6

1.0

0.0

0.8

Log-Rank

22 = 17.1020

p=0.0002

1: PB + / BM +

2: PB - / BM +

3: PB - / BM -

FCR-based therapy

~3 yrs median follow-up


Type of Therapy

Rawstron AC, et al. Haematolgica 2015; 100(S1):Abstract S794 (oral presentation) Data updated June 2016.

PB M‘D . %

Months after randomisation

3M

post-treatment

MRD

12 18 24

BM <0.01%

PB <0.01%

2.0%

3/148

6.0%

8/133

9.7%

12/124

BM >0.01%

PB <0.01%

33%

13/39

62%

18/29

81%

17/21




Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

0.2

0.4

0.6

0.8

1.0

0.0

6 12 18 24 30 36 60 0 42 48 54

Log-Rank

22 = 112.565

1: BM + / PB +

2: BM + / PB -

3: BM - / PB -

FCR-based therapy



Type of Therapy

Rawstron AC, et al. Haematolgica 2015; 100(S1):Abstract S794 (oral presentation); Data updated June 2016.



FCR-based therapy



Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

0.2

0.4

0.6

0.8

1.0

0.0

6 12 18 24 30 36 60 0 42 48 54

Log-Rank

22 = 112.565

1: BM + / PB +

2: BM + / PB -

3: BM - / PB -

FCR-based therapy

4 yrs median follow-up


Pro

po

rtio

n A

liv

e a

nd

Pro

gre

ssio

n F

ree

0.2

0.4

0.6

0.8

1.0

0.0

6 12 18 24 30 36 60 0 42 48 54 66

Log-Rank

22 = 153.742

p<0.0001

1: BM + / PB +

2: BM + / PB -

3: BM - / PB -

Compartment effect


• PB MRD <0.01%: patient may have extensive disease during or shortly after antibody therapy

• It is possible to assess the compartment effect per treatment

• Alemtuzumab: 2-log higher MRD levels in BM than in PB

• Rituximab: 0.7-log higher MRD levels in BM than in PB

• Ibrutinib/Idelalisib: no compartment effect

• Ibrutinib+Obinutuzumab: 0.3-log higher MRD levels in BM > PB

• PB is acceptable for monitoring (during and after therapy) but

BM may be required if there is a significant compartment effect

• PB >1% BM does not provide additional information

• Know the limit of detection (background) and limit of quantitation (sensitivity)

• FLOW: LoD 20 events, LoQ 50 events. Total events 2 million LoD 10-5

• PCR/HTS: 500ng 105 cells

• Factors that may impact individual assay results

• FLOW: Non-typical phenotype or treatment-associated phenotypic shift/drift

• PCR/HTS: Amplification bias

• Compartment effect: hemodilute samples

• FLOW: assess BM-restricted cell populations within the assay

• PCR/HTS: flow/morphology on paired sample

MRD in CLL

Acknowledgements Johan Dobber, Arnon Kater Academic Medical Center, Amsterdam, The Netherlands.

Remi Letestu, Florence Cymbalista AP-HP, Hôpital Avicenne, Bobigny, France

Martin Spacek Charles University in Prague, Czech Republic

Neus Villamor, Julio Delgado Hospital Clínic, Barcelona, Spain.

Josep Nomdedeu, Carol Moreno Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Claudia Fazi, Paolo Ghia Università Vita-Salute San Raffaele, Milan, Italy

Gabriele Brachtl, Alexander Egle Paracelsus Medical University, Salzburg , Austria.

Stuart Liptrot, David O'Brien St. James's Hospital, Dublin, Ireland.

Andy C. Rawstron, Ruth M. de Tute, Peter Hillmen St. James's Institute of Oncology, Leeds Teaching Hospitals, UK.

Olga Stehlikova, Michael Doubek, Sarka Pospisilova University Hospital Brno, Czech Republic.

Karl-Anton Kreuzer, Michael Hallek University of Cologne, Köln, Germany

Carlos Palacio, Francesc Bosch Vall d'Hebron Hospital, Barcelona, Spain.

Peter Gambell, David Westerman Peter MacCallum Cancer Centre, East Melbourne, Australia

Asha Soosapilla, Stephen Mulligan Royal North Shore Hospital, University of Sydney, Australia

Constance Yuan, Maryalice Stetler-Stevenson Center for Cancer Research, National Institutes of Health, Bethesda, MD, USA.

Gerard Lozanski Department of Pathology, The Ohio State University

Curtis Hanson, Tait Shanafelt Mayo Clinic, Rochester, Minnesota, USA.

Jeffrey L. Jorgensen, William G. Wierda MD Anderson Cancer Center, University of Texas

Beth Broome, Thomas J. Kipps Moores Cancer Center, University of California, La Jolla, CA, USA.

Fiona Craig University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Frans Nauwelaers, Lucia Testolin, Jingyi Chen and Noel Warner. BD

Biosciences provided custom and commercial conjugates for testing

Carol Moreno, EMILI MONTSERRAT and PAOLO GHIA

HTS vs. Sanger: Andreas Agathangelidis

Dilution studies: Claudia Fazi

Ruth de Tute, ANDREW JACK and PETER HILLMEN

Michael Wenger for meeting support

Comparison of flow vs. next

generation sequencing

Acknowledgements

NCRI National

Cancer

Research

Institute

Peter Hillmen (Chair)

David Allsup

Garry Bisshopp

Adrian Bloor

Daniel Catovsky

Claire Dearden

Caroline Duncan

Martin Dyer

Chris Fegan

George Follows

Helen McCarthy

Mel Oates

Piers Patten

Andy Pettitt

Chris Pocock

Guy Pratt

Anna Schuh

Jon Strefford

Renata Walewska

Nick York

NCRI CLL Trials Sub-group

Leeds CTRU

Anna Hockaday Dena Howard Jamie Ougton Lucy McParland Seoha Shanu Laura Collett Claire Dimbleby David Stones David Philips Sadia Aslam Kathryn McMahon James Baglin Walter Gregory Julia Brown

HMDS, Leeds Andy Rawstron Talha Munir

Abraham Varghese Ruth de Tute

Jane Shingles Cathy Burton

Francesco Forconi

Chris Fox

John Gribben

S Hewamana

Anna Hockaday

Dena Howard

Claire Hutchinson

Ben Kennedy

Scott Marshall

Alison McCaig

Janssen Novartis

Gilead Pharmacyclics

Roche

Bloodwise TAP Programme Rebecca Bishop Frankie Yates Kristian Brock Samuel Muñoz-Vicente Christina Yap Sonia Warner Shamyla Siddique

UKCLL Trials

Biobank, Melanie Oates

Melanie Goss

Emily Cass

Andy Pettitt

Liverpool CTU Matthew Bickerstaff James Dodd Lucy Read Chantelle Murphy Zelicia Gerald Jo Goulding Frances Sweeney Zviwone Gore Kate Culshaw Be adette O Do ell

Napp

Abbvie

I only want

MRD on 700

patients at 20

ti e poi ts…

http://www.ncri.org.uk/home/index.cfm

https://www.google.co.uk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=https://www.liv.ac.uk/party2015/keynote-speakers/&ei=Up5sVcrIC8XX7QadqYHwDg&bvm=bv.94455598,d.ZGU&psig=AFQjCNHPnqIY9TnWPYjp7feNNdyK-dPAxw&ust=1433268175533648

http://www.ox.ac.uk/

Flow cytometry for MRD detection

Key gating points for CLL MRD detection

“tart with CLL regio s i the e tre of the normal B-cell population

Adjust regions: CLL CD81/CD79b/CD20 lower than median of normal B-cells

Progenitors and plasmablasts have strong CD43/CD81 and no CD5

Contaminating T-cells have strong CD43/CD81 and (usually) no CD20

Contaminating T-cells usually removed by adjusting B-cell gate

CLL cells have similar CD19 expression to normal B-cells

Exclude events that are not fully resolved from normal B- ells y ≥1 para eter

Not CLL


??


CLL

Be very cautious around the limit of detection

CLL events n=51

Be very cautious around the limit of detection

Not CLL events

mrd in cll - ericll.org · fewer than one cll cell per 10,000 leukocytes •the blood can generally...

Documents