MRD in different disorders - CDDFcddf.org/files/2017/10/Session-4-Andy-Rawstron.pdf · MRD in different disorders Andy C. Rawstron ... requires a quantitative method that is not influenced

MRD in different disorders

Andy C. Rawstron

HMDS

St. James’s Institute of Oncology

Leeds Teaching Hospitals NHS Trust

MRD in CLL as an intermediate endpoint for licensure: requires a quantitative method that is not influenced by the

polyclonal background with prospective validation

www.ema.europa.eu/docs/en_GB/document_library/Scientific_guidel

ine/2014/12/WC500179047.pdf

Development of ‘MRD’ as aregulatory endpoint:

Identify MRD endpoint in clinical trials

Develop assay

Standardisation of assay

Apply standardised assay prospectively

Apply to regulatory action

1995

2007

2012

2002

B, bendamustine;C, Cyclophosphamide;

F, Fludarabine;M, mitoxantrone;

R, rituximab* Clinical responses only.

Chlorambucil2–4

90

80

70

50

30

20

10

0

60

40

Patie

nts(

%)

MRD-positive CR (or sample not available)

MRD-negative CR

Fludara-bine*3–5

Alem

tu-

zum

ab2 FC3,5,7,8

FCR7,

8

FCM

9

RFCM

10

BR6

1970 1980 1990 2000 2010 2020

MorphologyQualitativeLOD 1-10%

2/3-CLR Flow or IgH-PCRQualitativeLOD 0.1-1%

ERIC Flow RQ-PCR-CLR Quantitative

LOD 10-5 – 10-4

HTSQuantitative

LOD 10-6 – 10-5

VR11

1. Adapted from: Ghia P. Hematology 2012; 2012:97–104; 2. Hillmen P, et al. J Clin Oncol 2007; 25:5616–5623; 3. Catovsky D, et al. Lancet 2007; 370:230–239; 4. Eichhorst BF, et al. Blood 2009; 114:3382–3391; 5. Eichhorst BF, et al. Blood 2006; 107:885–891; 6. Fischer K, et al. J Clin Oncol 2012; 30:3209–3216; 7. Hallek M, et al. Lancet 2010; 376:1164–1174; 8. Böttcher S, et al. J Clin Oncol2012; 30:980–988; 9. Bosch F, et al. Clin Cancer Res 2008; 14:155–161; 10. Bosch F, et al. J Clin Oncol 2009; 27:4578–4584; 11. Seymour J, et al. Lancet Oncol 2017; 18:230-240.

Venetoclax (BH3-mimetic) can effect MRD-negative remissions: treatment cessation is a possibility

Andrew Roberts et al, N Engl J Med. 2016 Jan 28;374(4):311-22http://learningcenter.ehaweb.org/eha/2016/

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

Time (Years)

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.

2 4 6 8 10

0.01

0.1

1

10

100

0.001

0.0001

Ab

solu

te N

um

be

r o

f C

LL C

ells

(1

09/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 Survivalby bone marrow MRD level at 3 months post treatment

N=343 ×××××××

××

×××

×××

××××

×××

×××

××××

××

×

×××××××

×

××××××××××××

×××××××××××××××××××××××

××××

××××××××××××

×

×

× × ××

×

×

×

×

××××

×

×

×

<0.01%<0.1%<1%<10%≥10%

Log-Rank2

4 = 224.125p<0.0001

0 6 12 18 24 30 36 42 48 54 60

1.0

0.8

0.6

0.4

0.2

0.0Pro

po

rtio

n A

live

an

d P

rogr

ess

ion

Fre

e

Improving 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

LoD, limit of detection.

.

Markers requested for inclusion in cytometric MRD

analysis

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 ≤20% 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.

Requires ≥6 markers to achieve 0.01% –

available to most labs

The core panel must meet these 6 specifications, but

is flexible thereafter

Leeds routine: CD19 gating + core + ROR1 & CD23?CD19 loss: HLADR, ROR1 and CD19 gating + core (CD22 instead of CD20)

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

Requires ≥6 markers to achieve 0.01% –

available to most labs

The core panel must meet these 6 specifications, but

is flexible thereafter

Guideline MRD (<10-4)Measure to MRD5 (≤10-5) Explore HTS MRD6 (10-6)

How to report MRD responseCR with MRD >1% equivalent to PR

CR 4

CR 3CR 2

CR 1

CR

PR

SD

MRD 4 = <0.01% 1 in 10K (< 10-4) MRDMRD 5 = <0.001% 1 in 100K (< 10-5) MRDMRD 6 = <0.0001% 1/million (< 10-6) MRD

Developing a Mathematical Model Based on Partitions of Residual Disease Assuming a Lognormal Distribution:

? “Cure” Target ?

Gregory W, et al. XVI iwCLL Annual Meeting 2015.

Log

of

Re

sid

ual

Tu

mo

ur

(% M

RD

)

0.05 0.10 0.15 0.20 0.25 0.30

Probability Density

IWCLL threshold

Detection limit

• Ensuring broad access to techniques for measuring MRD and identifying suitable approaches to reporting response

Insights into MRD from CLL

Compartment effect: PB vs. BM

0.001

0.01

0.1

1

10

100

0.001 0.01 0.1 1 10 100

PB

CLL

% o

f le

uco

cyte

s

BM CLL % of leucocytes

Log Difference

Median >0.88Small -1.3Large >2.45

n total 272

BM+PB- n 59 (21.7%)

>2log diff 8 (2.9%)

Rituximab + Chemotherapy (3M post end Rx)

PFS for PB-BM+ PFS similar to PB-BM- in the short term

~3 yrs median follow-up

Months from Randomisation

Pro

po

rtio

n A

live

an

d P

rogr

ess

ion

Fre

e

0.2

0.4

0.6

0.8

1.0

0.06 12 18 24 30 36 600 42 48 54

Log-Rank2

2 = 112.565

1: BM + / PB +2: BM + / PB -3: BM - / PB -

Months from Randomisation

Pro

po

rtio

n A

live

an

d P

rogr

ess

ion

Fre

e

0.2

0.4

0.6

0.8

1.0

0.06 12 18 24 30 36 600 42 48 54 66

Log-Rank2

2 = 153.742p<0.0001

1: BM + / PB +2: BM + / PB -3: BM - / PB -

Compartment effect: PB vs. BM

0.001

0.01

0.1

1

10

100

0.001 0.01 0.1 1 10 100

PB

CLL

% o

f le

uco

cyte

s

BM CLL % of leucocytes

Log Difference

Median >0.88Small -1.3Large >2.45

n total 272

BM+PB- n 59 (21.7%)

>2log diff 8 (2.9%)

Rituximab + Chemotherapy (3M post end Rx)

PFS for PB-BM+ PFS worse than PB-BM- in the longer term

~4 yrs median follow-up

Compartment effect

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

• 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: BM ≥2.4 log higher than PB, 52% BM+PB-

• FC+Rituximab: BM ≥ 0.9 log higher than PB, 22% BM+PB-

• Ibrutinib/Idelalisib: BM = PB

• Ibr+Rituximab BM ≥ 0.2 log higher than PB, 6% BM+PB-

• Ibrutinib+Venetoclax BM ≥ 0.3 log higher than PB, 10% BM+PB-

• Ibr+Obinutuzumab BM ≥ 0.5 log higher than PB, 9% BM+PB-

• PB is acceptable for monitoring (during and after therapy) but BM may be required if there is a significant compartment effect

• BM is required for definitive MRD response assessment

Compartment effect in MM

Br J Haematol. 1997 Apr;97(1):46-55


• Understanding compartment effects


Kinetics of response to BCR-pathway inhibitors

№ o

f p

atie

nts

>5%

0.5-5%

<0.5%

CLL cells % Ki67+

Fo

ld C

han

ge

in

per

iph

eral

B-

cell

cou

nt

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

Rapid (4-24hrs) entry of proliferating cells into blood. Peripheral counts peak at week 1 as proliferation

starts to decline

№ o

f p

atie

nts

>5%

0.5-5%

<0.5%

CLL cells % Ki67+

Fo

ld C

han

ge

in

per

iph

eral

B-

cell

cou

nt


Rapid (4-24hrs) entry of proliferating cells into blood. Peripheral counts peak at week 1 as proliferation

starts to decline

№ o

f p

atie

nts

>5%

0.5-5%

<0.5%

CLL cells % Ki67+

Fo

ld C

han

ge

in

per

iph

eral

B-

cell

cou

nt


The dynamic cellular kinetics of chronic lymphocytic leukemia B cells

The birth and death rates of CLL cells vary between patients

Brad Messmer et al, J Clin Invest. 2005 Mar;115(3):755-64

Measuring the kinetics of response to BCR-pathway inhibitors: MRD analysis to determine

“CLL halving-time”

0.001

0.01

0.1

1

10

100

1000

0 6 12 180.001

0.01

0.1

1

10

100

1000

1 2 6 9 12 18

Ab

solu

te C

LL C

ell

Co

un

t(1

09/L

)

Months of Ibrutinib Treatment




0.001

0.01

0.1

1

10

100

1000

0 6 12 180.001

0.01

0.1

1

10

100

1000

1 2 6 9 12 18

Ab

solu

te C

LL C

ell

Co

un

t(1

09/L

)





0.001

0.01

0.1

1

10

100

1000

0 6 12 18

Ab

solu

te C

LL C

ell

Co

un

t(1

09/L

)



0.001

0.01

0.1

1

10

100

1000

1 2 6 9 12 18



0.001

0.01

0.1

1

10

100

1000

0 6 12 18

Ab

solu

te C

LL C

ell

Co

un

t(1

09/L

)



Exponential depletion unless plateau reached

Clonal evolution leading to ibrutinib resistance in chronic lymphocytic leukemia

Inhye E. Ahn et al Blood 2017 129:1469-1479; doi: https://doi.org/10.1182/blood-2016-06-719294

Blood CLL Responses

- log scale

The rate of fall is rapid in all

patients with a median of 3 log

reduction in CLL level after 8

weeks of combined therapy.

Hillmen et al., Poster, IWCLL, 2017, Board 421

Individual responses to ibrutinib + venetoclax

<0.001

0.001

0.01

0.1

1

10

100

1000

0 12 24 36 48 60<0.001

0.001

0.01

0.1

1

10

100

1000

0 12 24 36 48 60

<0.001

0.001

0.01

0.1

1

10

100

1000

0 12 24 36 48 60<0.001

0.001

0.01

0.1

1

10

100

1000

0 12 24 36 48 60

Time (weeks)

CLL

cel

l co

un

t (1

09/L

)

CLL

cel

l pe

rcen

tage

of

leu

cocy

tes

Rapid log-linear reduction

Slow log-linear reduction

? MRD plateau at MRD5 ?

MRD plateau

1%

Stopping treatment in UK trials: change from 6M after confirmed MRD4 to double the time to achieve MRD4

Log / month

On Rx (mths)

Rx-free(mths)

Total (mths)

0.3 22 36 58

0.6 14 48 62

1.0 12 72 84

Log / month

On Rx (mths)

Rx-free(mths)

Total (mths)

0.3 32 56 88

0.6 18 64 82

1.0 12 72 84

1E-08

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

100

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90

CLL

cel

l co

un

t (1

09 /

L)

Time (months)

Time until disease progression (lymph count > 5 x 109/L) in months, assuming stable doubling time of 2 months from end of treatment

Stop Rx 6M after confirmed MRD <0.01% Stop Rx after double time to MRD <0.01%



• Improving knowledge of MRD kinetics to optimise treatment


Using MRD techniques to measure pro- & anti-apoptotic protein expression during treatment

Baseline

8wks ibrutinib(pre-venetoclax)

8M ibrutinib6M venetoclax

For example only – not necessarily representative, analysis ongoing

Using MRD techniques to measure pro- & anti-apoptotic protein expression during treatment

8M ibrutinib6M venetoclax


CLL

B-progenitors

Inhibiting BCR signalling

Zenz T, Nature Reviews Cancer 2010, 10:37-50

Herman SE et al Leukemia. 2013 Dec;27(12):2311-21

Measuring protein phosphorylation in residual disease during inhibitor treatment

Fold

incr

ease

in m

edia

n f

luo

resc

ence

in

ten

sity

rel

ativ

e to

un

stim

ula

ted

cel

ls

Syk P348 Akt S473

Treatment naive

Relapsed refractory

Talha Munir, ASH 2016

Measuring protein phosphorylation in residual disease during inhibitor treatment

Surita Dalal

8wks ibrutinibPre-venetoclax

14wks ibrutinib6wks venetoclax

No stimulus

BCR-stimulus (IgM + IgD)




• Improving knowledge of MRD kinetics to optimise treatment

• Identifying the biological response at an MRD level


Acknowledgements

NCRINational

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 CTRUAnna Hockaday Dena HowardJamie Oughton Fabrizio MessinaLucy Williams Lucy McParlandClaire Dimbleby David StonesDavid Philips Chris LinseySeren Langley David CairnsJames Baglin Walter GregoryJulia Brown

HMDS, LeedsAndy 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

GileadPharmacyclics

Roche

Bloodwise TAP ProgrammeRebecca Bishop Francesca YatesKristian Brock Samuel Muñoz-VicenteTina McLeod Sonia foxYolande Jeffferson-Hulme Sophie CrampShamyla Siddique Charles Craddock

UKCLL Trials

Biobank, Melanie Oates

Melanie Goss

Emily Cass

Andy Pettitt

Liverpool CTUMatthew BickerstaffJames DoddLucy ReadChantelle MurphyZelicia GeraldJo GouldingFrances SweeneyZviwone GoreKate Culshaw Bernadette O’Donnell

Napp

Abbvie Acerta/AZ

University of LeedsKatie Holmes Surita Dalal

Nicola McWhirter Alasdair Dewar

Pascal LeFevre Darren Newton

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

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

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

Documents

MRD in different disorders - CDDFcddf.org/files/2017/10/Session-4-Andy-Rawstron.pdf · MRD in different disorders Andy C. Rawstron ... requires a quantitative method that is not influenced