mature b-cell lymphoproliferative disorders - escca · mature b-cell lymphoproliferative disorders...

23/10/2017

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Mature B-cell lymphoproliferative disorders

Andy C. Rawstron

HMDS

St. James’s Institute of Oncology

Leeds Teaching Hospitals NHS Trust

DISCLOSURES OF COMMERCIAL SUPPORT

Name of

Company

Research

support

Employee Consultant Stockholder Speaker’s

Bureau

Advisory

Board

Other

Abbvie X X

BD Bio-sciences

reagents X

Beckman Coulter

reagents X

Celgene X X X

Gilead X X X

Janssen X X

Pharma-cyclics

X

Roche X X

Chronic Lymphocytic Leukaemia / Small Lymphocytic Lymphoma

• Incidence 7.1 per 100K per year

• Abnormal B-cells in the blood (>5 x 109/L), bone marrow and or tissues

• B-cells co-express CD19, CD5 and CD23 with weak sIg, CD79b, CD20, CD22.

• Treatment depends on clinical features: cytopenia, rate of progression, lymphadenopathy, ~85% do not require treatment at presentation in UK

• Precursor syndrome MBL (<5 x 109/L) incidence 2.6/ 100K/year: ~1% progression to CLL per year

• “low-count” MBL (<0.5 x 109/l) – no known clinical consequences

www.hmrn.org.uk

<60

60-69

70-79

>80

Survival relative to age- and sex-matched general population

The genomic landscape in CLL

Puente XS et al, Nature 2015; 526[7574]: 519-24.Sander et al Haematologica. 2008;93(5):680-7

13q14 deletion is the most common

abnormality in CLL but also other in disorders,

e.g. ~10% of mantle cell lymphoma

Pathway analysis of inherited susceptibility loci show a role for GC-transition & apoptosis

Sonja Berndt et al 2016 Mar 9;7:10933. doi: 10.1038/ncomms10933.

Rawstron et al, Lancet Haematology Volume 4,

No. 7, e334–e340, July 2017

IGHV mutation status and BCR signalling

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

~30% of patients with CLL carry immunoglobulin receptors with highly similar primary sequence

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The Unique IgH Gene Sequence

VH1 VH2 VHx D1 D2 Dy JH1 JH2 JHz

VHx DyJHz

+ P - looping of

complimentary nucleotides

onto ss signal joint

+ N - 3’ insertion of 1-20

nucleotides by TdT

- Exonuclease trimming of

unpaired nucleotides

Bone Marrow:

IGHV re-arrangement

C

Lymph nodes:

Affinity Maturation

Features associated with oncogenesis vs. disease progression

CLL-type MBL

(clinic MBL)

CLL cells:

0.1-4.9 x 109/L

CLL-like B-cells

(low-count MBL)

CLL cells:

<0.1 x 109/L

PhenotypeCD19+CD5+CD23+

CD20wkCD79b/sIgwk

CD19+CD5+CD23+

CD20wkCD79b/sIgwk

Inherited pre-

disposition SNPAs CLL As CLL

Chromosomal

Abnormalities

13q14 deletion common;

11q23/17p deletions rare

13q14 deletion in some cases;

11q23/17p deletions v. rare

IGHV repertoire

Similar to CLL

(most common: IGHV 3-07,

1-69, 4-34, 3-23)Different to CLL

BCR pathway inhibitors in CLL: high response rates & durable remissions

Burger et al, N Engl J Med. 2015 Dec 17; 373(25): 2425–2437

Simplified overview of potential B-cell-receptor-mediated events related to antigen- and superantigen-binding in CLL.

Bühler A et al. Clin Cancer Res 2010;16:373-375

e.g. non-muscle myosin heavy

chain IIA*

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling

Dühren-von Minden M et al Nature. 2012 Sep 13;489(7415):309-12.

(a) The Fab molecules in both the CLL183 and CLL240 crystals interact through VH CDR3-dominated contacts. The heavy and light chains of the Fab acting as ‘receptor' are coloured lightblue and pink, with the VH CDR3 loop highlighted in red. The chains in the ‘antigen' molecule arecoloured blue and cyan (VH and CH1 domains), and violet and magenta (VK and CK), respectively. (b)Molecular surface of the epitope, with the amino acids from VH CDR3 involved in the bindinginteractions shown as sticks coloured as in the previous panel.

Distinct homotypic B-cell receptor interactions in CLL

Minici et al. Nat Commun. 2017; 8: 15746

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Mechanisms and clinical impact of Bcl-2 dysregulation in CLL

• 13q14 deletion: • miR-15/miR-16 family

• Downregulate BCL2 gene expression del13q14 leads to Bcl-2 over-expression.

• Inherited Susceptibility• Genetic polymorphisms that

impact on BCL2 family

• Trisomy 12• Lower bax/bcl-2 ratio

• BLC2-IGH translocation

Clinical Significance Of Bax/Bcl-2 Ratio In CLLMaria Ilaria Del Principe et al

Haematologica January 2016 101: 77-85

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/

• CLL: no driver mutation, expansion driven by BCR-signalling (possibly autonomous) coupled with reduced propensity for apoptosis

Summary

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.

MRD is an independent predictor of outcomeCR with MRD >1% equivalent to PR

CR 4

CR 3CR 2

CR 1

CR

PR

SD

MRD 1 = <10% 1 in 10 (< 10-1) MRDMRD 2 = <1% 1 in 100 (< 10-2) MRDMRD 3 = <0.1% 1 in 1K (< 10-3) MRDMRD 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

Impact of MRD on long-term survival

Rawstron et al. Blood 2001;98:29-35

Publication-quality graphic from 2001!

Marwan Kwok et al. Blood 2016;128:2770-2773

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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

Stopping treatment in UK trials: initial plan to stop 6M after confirmed MRD4 (<0.01%)

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

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% Ibrutinib costs £55K/patient/yrUK NHS accepts up to ~£30K/yr

Funded for relapsed/refractory or 17p del/mut TN but concerns about side effects, selection of resistant clones and high cost UK trials aimed at stopping therapy

PB monitoring until 6M sustained MRD4 (<0.01%) confirmed in BM.

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)

MRD as an endpoint needs a highly validated quantitative assay


• 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 qPCR• Validated assays require “limit of detection” for each result

stated on the report• Cellular assays: number of events acquired• Molecular assays: total DNA, patient-specific IGH effects

• Combination of both ≥6-CLR flow cytometry and HTS is optimal

• Should MRD assessment be performed in PB or BM?

Alemtuzumab +/- Chemotherapy (during/end Rx)

Log Difference

Median >2.44Small -1.3Large >4.34

n total 142

BM+PB- n 74 (52.1%)

>2log diff 71 (50%)

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

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

Type of Therapy

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

PFS for PB-BM+similar to PB+BM+

Alemtuzumab therapy

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

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 -


Pro

po

rtio

n A

live

an

d P

rogr

ess

ion

Fre

e

6 12 18 24 30 36 600 42 48 54

0.2

0.4

0.6

1.0

0.0

0.8

Log-Rank2

2 = 17.1020p=0.0002

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

FCR-based therapy~3 yrs median follow-up

Rituximab + Chemotherapy (3M post end Rx)

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


Log Difference

Median >0.88Small -1.3Large >2.45

n total 272

BM+PB- n 59 (21.7%)

>2log diff 8 (2.9%)

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

Type of Therapy

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

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

FCR-based therapy~3 yrs median follow-up


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 -

FCR-based therapy4 yrs median follow-up


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 -

Ibrutinib / Idelalisib monotherapy (1M/6M Rx)

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


Log Difference

Median >0Small -0.72Large >1.1

n total 111

BM+PB- n 0 (0%)

>2log diff 0 (0%)

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


IcX: Ibrutinib + Obinutuzumab (1M/9M 3 months post obinutuzumab)

Log Difference

Median >0.46-0.36

Large >2.74

n total 55

BM+PB- n 5 (9.1%)

>2log diff 5 (9.1%)

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: no compartment effect

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

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

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

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• BCR/BCL2-pathway inhibitor combinations are effecting deep remissions and MRD monitoring is being used to guide treatment duration

Summary CLL – treatment over time

Kinetics of response to BCR-pathway inhibitors

№ o

f pat

ien

ts

>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№

of 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 pat

ien

ts

>5%

0.5-5%

<0.5%

CLL cells % Ki67+

Fo

ld C

han

ge

in

per

iph

eral

B-

cell

cou

nt


Loss of normal proliferating CLL cell expression profile during ibrutinib therapy

CX

CR

4

Ki-67 expression

CX

CR

4

CX

CR

4

Ki67Ki67CXCR4 requires BTK for internalisation and signalling.

Ibrutinib rapid loss of homing to proliferation centres

Ibrutinib

1 month

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The dynamic cellular kinetics of chronic lymphocytic leukemia B cells

The birth and death rates of CLL cells vary between patients

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

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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 per

cen

tage

of

leu

cocy

tes

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 16 56 72

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 96

CLL

cel

l co

un

t (1

09 /

L)

Time (months)

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% • CLL: no driver mutation, expansion driven by BCR-signalling (possibly autonomous) coupled with reduced propensity for apoptosis


• CLL cells have a birth rate, potentially abrogated by BCR-pathway inhibition, and a death rate, potentially enhanced by BCL2-pathway inhibition exponential decline in disease until plateau

Summary

Inhibiting BCR signalling

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

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

CLL cell %+ CLL median CLL fold change

No stimulus 2.42 0.31 1.00

Inhibitor 1.46 0.22 0.71

IgM/D 44.08 1.04 3.35

Inhibitor + IgM/D 24.18 0.72 2.32

No inhibitorNo stimulus

InhibitorNo stimulus

InhibitorIgM/D

No inhibitorIgM/D

Analysis of CLL Syk phosphorylation: ? Unsuitable even as exploratory endpoint ?

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No inhibitorNo stimulus

InhibitorNo stimulus

InhibitorIgM/D

No inhibitorIgM/D

Analysis of CLL and T-cell Syk phosphorylation: Analysis of CLL and T-cell Syk phosphorylation:

CLL cell %+

CLL median

CLL fold change

T-cell median

T-cell fold change

CLL/T-cell MFI ratio

No stimulus 2.42 0.31 1.00 0.30 1.00 3.33

Inhibitor 1.46 0.22 0.71 0.33 1.10 2.15

IgM/D 44.08 1.04 3.35 0.27 0.90 12.4

Inhibitor + IgM/D 24.18 0.72 2.32 0.31 1.03 7.48

Fold change in phospho-Syk/Akt after Ig stimulation in CLL cells from patients on ibrutinib

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




• Potential efficacy of inhibitor therapies readily & rapidly assessable using Phosflow in vitro for drug development and in vivo for clinical trials

Summary

Lymphoplasmacytic lymphoma / WaldenstromMacroglobulinaemia – simple model.

• B cell component

• Symptoms related to tumour bulk

• Anaemia

• B symptoms

• Lymph nodes

• Spleen

• Plasma cell component

• Symptoms attributable to M-protein

• Hyperviscosity syndrome

• Neuropathy

• Haemolytic anaemia

• Cryoglobulinaemia

• Immunodeficiency

MYD88 L265P: driver mutation in the majority of LPL/WM and IgM MGUS

• MYD88 mutation:

• spontaneous homodimerization and recruitment of IRAK1/4

• promotes NF-κB also by activating the Bruton's tyrosine kinase (BTK)

Davide Rossi Hematology2014;2014:113-118

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10

2012

2015

Treon et al, NEJM; 2015

Response to ibrutinib associated with CXCR4 mutation status

Mantle cell lymphoma – first line treatment

HMRN real world data

MCL – second line treatment over time

HMRN real world data

23/10/2017

11




• Potential efficacy of inhibitor therapies readily & rapidly assessable using Phosflow in vitro for drug development and in vivo for clinical trials

• Btk-inhibition also effective in MYD88-mutated disorders, potentially modulated by capacity for developing resistance mutations, and has promising activity in mantle cell lymphoma

SummaryAcknowledgements

NCRINational

CancerResearch

Institute

Peter Hillmen (Chair) David AllsupGarry BisshoppAdrian BloorDaniel CatovskyClaire DeardenCaroline DuncanMartin DyerChris FeganGeorge Follows

Helen McCarthyMel OatesPiers PattenAndy PettittChris PocockGuy PrattAnna SchuhJon StreffordRenata WalewskaNick York

NCRI CLL Trials Sub-group

Leeds CTRUAnna Hockaday Dena HowardJamie Ougton Lucy McParlandSeoha Shanu Laura CollettClaire Dimbleby David StonesDavid Philips Sadia AslamKathryn McMahon James BaglinWalter Gregory Julia Brown

HMDS, LeedsAndy Rawstron Talha MunirAbraham Varghese Ruth de TuteJane Shingles Cathy Burton

Francesco ForconiChris FoxJohn GribbenS HewamanaAnna HockadayDena HowardClaire HutchinsonBen KennedyScott MarshallAlison McCaig

Janssen Novartis

GileadPharmacyclics

Roche

Bloodwise TAP ProgrammeRebecca Bishop Frankie YatesKristian Brock Samuel Muñoz-VicenteChristina Yap Sonia Warner

Shamyla Siddique

UKCLL TrialsBiobank,

Melanie Oates Melanie GossEmily CassAndy Pettitt

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

Napp

Abbvie

I only want MRD on 700

patients at 20 time points…

CD22 vs. CD20: B-cell CD20 is lost but CD22 expression persists during anti-CD20 therapy

Rawstron et al. Blood 2001;98:29-35ERIC CLL MRD Leukemia 2016;30(4):929-936

CD22 does not improve MRD detection in steady state over CD19,

CD5, CD20, CD43, CD79b & CD81

CD20 provides much better separation of CLL cells from normal

B-cells than CD22 in steady state

CLL

vs.

B s

epar

atio

n

Best

Worst

CD20

CD22

BCR-pathway inhibitor

Before treatment 1 month 6 months

BCR inhibitor with anti-CD20

Before anti-CD20(14 months BCRi)

1 week anti-CD20(15 months BCRi)

6 months anti-CD20(20 months BCRi)

High-Throughput Sequencing Shows GoodLinearity to One CLL Cell in One Million Leukocytes


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

0.000010.00001 0.0001 0.001 0.01 0.1 1 10

CLL

% L

eu

kocy

tes

Usi

ng

Ad

apti

ve C

lon

oSE

Q

Expected CLL % Leukocytes

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-associated” sequences 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 ValidationUsing synthetic templates to design an unbiased multiplex PCR assay. Nat Commun. 2013;4:2680.

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

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

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/

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

23/10/2017

12

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

? “Cure” Target <<1 CLL Cell in 10 Million Leucocytes ?

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 0.30Probability Density

0500 billion1 trillion 102 104 106 108 1010 1012

Tumour burden

Log scale(MRD assay)

Linear scale(morphology)

PR

CR (MRD 10-3)

MRD 10-4

MRD 10-5

MRD 10-6

MRD 10-7

MRD 10-8

MRD 10-9

MRD 10-10

MRD 10-11

MRD 10-12

Morphological remission

MRD measurable

MRD not measurable but destined to relapse

MRD not measurable potentially operational

cure

~1yr ad

ditio

nal P

FS per lo

g dep

letion

CR/PR (1%)

Design and timing of assay is critical

Duration of stimulus may have significant impact

IgM+IgD stimulation optimal 2 minutes

Viability of cells is critical

Day 0 analysis not possible all analyses on Day 1

0

1

2

3

4

5

Day 0 Day 1 Day 2

TAP trial development: Talha Munir

Manne BK,J Biol Chem. 2015 May 1;290(18):11557-68. Syk protein activation downstream of ITAM/hemITAM receptors in platelets

Patel V et alClin Cancer Res. 2017 Jul 15;23(14):3734-3743.

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