mature b-cell lymphoproliferative disorders - escca · mature b-cell lymphoproliferative disorders...
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23/10/2017
1
Mature B-cell lymphoproliferative disorders
Andy C. Rawstron
HMDS
St. James’s Institute of Oncology
Leeds Teaching Hospitals NHS Trust
DISCLOSURES OF COMMERCIAL SUPPORT
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reagents X
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reagents X
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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
Rawstron AC, et al. Leukemia 2016; 30:929–936.
• 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 -
Months from Randomisation
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
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%)
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
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 -
FCR-based therapy4 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 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
BM CLL % of leucocytes
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
BM CLL % of leucocytes
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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• CLL: no driver mutation, expansion driven by BCR-signalling (possibly autonomous) coupled with reduced propensity for apoptosis
• 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
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 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
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
IcICLLe: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB
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
IcICLLe: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB
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 18
Ab
solu
te C
LL C
ell
Co
un
t(1
09/L
)
Months of Ibrutinib Treatment
IcICLLe: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB
0.001
0.01
0.1
1
10
100
1000
1 2 6 9 12 18
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 18
Ab
solu
te C
LL C
ell
Co
un
t(1
09/L
)
Months of Ibrutinib Treatment
IcICLLe: https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-003608-11/GB
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
• BCR/BCL2-pathway inhibitor combinations are effecting deep remissions and MRD monitoring is being used to guide treatment duration
• 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
• CLL: no driver mutation, expansion driven by BCR-signalling (possibly autonomous) coupled with reduced propensity for apoptosis
• BCR/BCL2-pathway inhibitor combinations are effecting deep remissions and MRD monitoring is being used to guide treatment duration
• 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
• 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
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• CLL: no driver mutation, expansion driven by BCR-signalling (possibly autonomous) coupled with reduced propensity for apoptosis
• BCR/BCL2-pathway inhibitor combinations are effecting deep remissions and MRD monitoring is being used to guide treatment duration
• 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
• 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
Rawstron AC, et al. Leukemia 2016; 30:929–936.
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.
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.