1

Steven P. Treon

Dana-Farber Cancer Institute

Harvard Medical School

Jan Gosta Waldenstrom

Described Macroglobulinemia in 1943

2nd Intl Workshop on Waldenstrom’s Macroglobulinemia

Athens 2002

WM Treatment Approach

WM Treatment Approach

NCCN

Options for

WM Treatment

CHLORAMBUCIL

CYCLOPHOSPHAMIDE

NUCLEOSIDE ANALOGUES

THALIDOMIDE

RITUXIMAB

EVEROLIMUS

CHOP-R, CVP-R, CPR, CDR

BORTEZOMIB (BDR, VR)

BENDAMUSTINE (BENDA R)

7

Agent WM Toxcities

Rituximab • IgM flare (40-60%)-> Hyperiscosity crisis, Aggravation of IgM related PN, CAGG, Cryos.

• Hypogammaglobulinemia-> infections, IVIG • Intolerance (15%)

Nucleoside Analogues

• Hypogammaglobulinemia-> infections, IVIG • Transformation, AML/MDS (15%)

IMIDS • Peripheral Neuropathy (60% >grade 2 with Thalidomide)

• Aggravated IgM flare (Revlimid and Pomalidomide)

• Severe anemia (Revlimid)

Bortezomib • Grade 2+3 Peripheral neuropathy (60-70%); High discontinuation (20-60%)

WM–centric toxicities with commonly used therapies

New Directions in WM

WHOLE GENOME SEQUENCING IN WM

Congratulations it only took you

70 years!!

NORM ≈≈≈≈≈≈≈≈≈≈≠≈≈

Paired Sequencing

from same individuals

WM ≈≈≈≈≈≈≈≈

3,000,000,000

nucleotides

MYD88 L265P Somatic Mutation

• Sanger sequencing (91%

of WM pts, 10% IGM MGUS

Have MYD88 L265P.

C to G at position 38186241

at 3p22.2 in 27/30 WM pts. Acquired UPD at 3p22.

Treon et al, NEJM 2012

MYD88 L265P in WM/IGM MGUS

METHOD TISSUE WM IGM MGUS

Treon WGS/Sanger BM CD19+ 91% 10%

Xu AS-PCR BM CD19+ 93% 54%

Gachard PCR BM 70%

Varettoni AS-PCR BM 100% 47%

Landgren Sanger BM 54%

Jiminez AS-PCR BM 86% 87%

Poulain PCR BM CD19+ 80%

Argentou PCR-RFLP BM 92% 1/1 MGUS

Willenbacher Sanger BM 86%

Mori AS-PCR/BSiE1 BM 80%

Ondrejka AS-PCR BM 100%

Ansell WES/AS-PCR BM 97%

Patkar AS-PCR BM 85%

11

MYD88 L265P by AS-PCR can help

distinguish WM from overlapping entities

WM

Xu et al, Blood 2013

WM

HD IGG IGM CLL MM MZL WM ----MGUS----

0% 0% 54% 4% 0% 10% 93%

13

MYD88 L265P N=36

MYD88 WT N=41

p=

BM (IHC) 9 (43%) 12 (57%) 0.04

IgM 686 (243-4728) 345 (116-1880)

<0.0001

IgA 141 (26-571)

194 (34-2520)

0.04

IgG 925 (82-3100)

1070 (206-3487) 0.04

Progression 6 WM (17%) 1 SMZL (3%)

1 WM (3%) 1 SMZL (3%)

NA

MYD88 Mutation Status in IGM MGUS

MYD88 L265P OR 4.7 (95% CI 0.8-48.7; p=0.04)

Varettoni et al, BLOOD 2013

Detection of MYD88 L265P using AS-PCR in Peripheral Blood

CD19-Selected Cells By Dynabead Magnetic Selection.

Xu et al, Leukemia 2014

Sensitivity of 98.2%, specificity of 100%,

positive and negative predictive values of

100% and 75%.

15

ΔCT of Peripheral Blood MYD88 L265P can help segway

IgM MGUS versus WM at time of diagnosis

Xu et al, Leukemia 2014

MYD88 L265P ΔCt was <6.5 in 82% of smoldering, and 94%

of symptomatic WM patients, whereas all IgM MGUS patients

had a ΔCt >6.5 (p<0.0001).

16

Copy number losses are common in WM

and effect growth and survival regulatory genes.

Hunter et al, Blood 2013

Generation of MYD88 L265P Conditional Transgenic Mice

Conditional

MYD88 LSL

Compound

MYD88 (L265P) LSL/LSL;AIDCre/+

AIDCre/+

MYD88

1. Hu-wt-MYD88

2. Hu-mutant-MYD88(L265P)

3. Mo-wt-MYD88

4. Mo-mutantt-MYD88(L26P)

MYD88

1. Vector Preparation

White coat

2. ESC electrophoration and selection

3. Blastocyst injection and

implantation into

pseudopregnant femele

Black coat 4. Generation Chymeric mice

5. Checking for germ line transmission

6. Generation of MYD88/AIDCre compound mice

Black/White coat

Removal of transcriptional stopper and expression in germinal centre B-cells

IWMF CANADA

MYD88

inhibitor

blocks p65

NFKB

nuclear

localization in

L265P

expressing

WM cells.

Control MYD88 Inhibitor

A.

B.

Treon et al, NEJM 2012 A. BCWM.1 B. MWCL-1

MYD88

TLR4

TIR

AP

BTK MYD88

IκBα

TRAF6

NEMO

IKKα IKKβ

p50 p65 Nuclear Translocation and Signaling

IL1R

MYD88 L265P

Signal Pathway

Yang et al, Blood 2013

NFKB

TAK1

A. B.

IB: MYD88

IgG - heavy chain

IB: BTK

IP:

IP:

BCWM.1 MWCL-1 OCI-Ly19 Ramos

IgG - heavy chain

IB: MYD88

IB: BTK

15.1 81.8 32.8 89.0 18.9 17.0 13.1 24.4 Ratio: MYD88 / BTK (%)

C.

IgG MYD88 IP:

IB: MYD88

IB: BTK

BCWM.1 MWCL-1 OCI-Ly3 Ramos RPMI-8226 OCI-Ly19

MYD88 binds to Bruton’s Tyrosine Kinase (BTK) in L265P expressing WM cells

Yang et al, Blood 2013 20

BCWM.1 MWCL-1

p-BTK

BTK

GAPDH

BCWM.1 MWCL-1

p-BTK

GAPDH

MYD88

BTK

100 60.8 100 71.2 relative density of phos-/total BTK (%)

p-BTK

Flag-MYD88

endogenous-MYD88

BTK

BCWM.1 MWCL-1

GAPDH

100 107 173 100 96.3 187 relative density of phos- / total BTK (%)

MYD88 L265P induces BTK in WM cells

Yang et al, Blood 2013 21

IBRUTINIB: Small Molecule Inhibitor of BTK

Binds to and inactivates BTK

Orally drug taken daily resulting in 24-hr BTK inhibition.

Watch for drug interactions

No coumadin

Well tolerated. Side effects also depend on prior therapies.

Blocks signaling that allows lymphoma cells to grow and survive.

Honigberg LA et al: Proc Natl Acad Sci U S A.107:13075, 2010

Herman SEM et al: : Blood. 2011 Mar 21. [Epub]

Ponader, et al., Proc ASH, 2010; Yang et al. Proc. ASH 2012.

N

N

N

N

N H 2

O

N

O

IBRUTINIB

Screening

Informed Consent and Registration

Ibrutinib

420 mg po daily

Progressive Disease or

Unacceptable Toxicity SD or Response

Continue x 26 cycles

Stop Ibrutinib

Event Monitoring

Event Monitoring

Opened May 2012 DFCI, MSKCC, STANFORD

N=35; expanded to 63

23

Phase II Study of Ibrutinib in Relapsed/Refractory WM

Baseline Characteristics for Study Participants (n=63)

Median Range

Age (yrs) 63 44-86

Male/Female 48/15 N/A

Prior therapies 2 1-8

Hemoglobin (mg/dL) 10.5 8.2-13.8

Platelet (k/uL) 214 24-459

Serum IgM (mg/dL) 3,610 735-8,390

B2M (mg/dL) 3.9 1.3-14.2

BM Involvement (%) 70 3-95

Adenopathy >1.5 cm 38 (60.3%) N/A

Splenomegaly >15 cm 7 (11.1%)

24 Data Lock: November 8, 2013

Reasons for Treatment Initiation

Anemia 55 (87.3%)

Adenopathy and/or Splenomegaly 16 (25.3%)

Neuropathy 10 (15.9%)

Hyperviscosity 6 (9.5%)

Thrombocytopenia 5 (7.9%)

Malignant Pleural Effusions 3 (4.7%)

Amyloidosis 2 (3.1%)

Acquired VWF deficiency 2 (3.1%)

Fatigue 2 (3.1%)

Nephropathy 1 (1.6%)

25 Data Lock November 8, 2013 (Multiple reasons can apply for a patient)

26

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

Baseline Cycle 2 Cycle 3 Cycle 6 Cycle 9 Cycle 12 Cycle 15

Serial Serum IgM Levels Following Ibrutinib

Data Lock November 8, 2013

N=63

Seru

m Ig

M (

mg

/dL)

Best IgM Response: 3,610 to 1,260 mg/dL; p=1.46033-17

Median of 9 (range 1-18) Cycles

8

10

12

14

16

Baseline Cycle 2 Cycle 3 Cycle 6 Cycle 9 Cycle 12 Cycle 15 Cycle 18

27

Serial Hemoglobin Levels Following Ibrutinib

N=63

Data Lock November 8, 2013

Best Hemoglobin Response: 10.5 to 13.4; p = 9.0366-19

Hem

ogl

ob

in (

g/d

L)

Median of 9 (range 1-18) Cycles

Bone Marrow Disease Burden following Ibrutinib

Data Lock November 8, 2013 28

BM

In

volv

emen

t (%

)

N= 61 (with baseline and cycle 6 BM Biopsy)

At Cycle 6: 62.5% to 36.75%; p = 0.00053

0

10

20

30

40

50

60

70

80

90

100

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61

Best Clinical Responses to Ibrutinib Median of 9 (range 1-18) Cycles

(N=) (%)

VGPR 6 9.5

PR 34 54

MR 12 19

• ORR: 83% Major RR (> PR): 64% • 87% on therapy at 9 months

Data Lock November 8, 2013

29 Response criteria adapted from 3rd International Workshop on WM (Treon et al, BJH 2011)

ORR 85.7%

Major (PR or better) 66.7%

Response by previous lines of treatment Response by relapse or refractory status

Relapse ORR 84% Major 63%

Refractory ORR 88% Major 72%

p=0.73 and 0.58 for ORR and Major RR

1 ORR 82% Major 59%

2-4 ORR 86% Major 66%

>4 ORR 92% Major 83%

p=0.81 and 0.37 for ORR and Major

RR

Updated investigator response assessments (May 2014)

Ibrutinib Related Adverse Events

31 Data Lock November 8, 2013

Adverse event # Grade 2

Events # Grade 3

Events # Grade 4

Events Total # of

Events

Neutropenia 3 7 2 12 Thrombocytopenia 3 6 1 10

Bleeding 4 1 5 Pneumonic Infection 2 1 3

Anemia 2 1 3 Tachyarrhythmia 2 1 3

Syncope/Pre-syncope 1 1 2 Herpes Zoster 1 1 2

Endocarditis infective 1 1 Fever 1 1

Hypertension 1 1 Pruritus 1 1

Mucositis 1 1 Oral Pain 1 1 Diarrhea 1 1

Arthralgia 1 1

At a median of 9 (range 1-18) cycles of ibrutinib:

55 (87.3%) participants continue on treatment. 8 participants discontinued protocol therapy:

• 1 Non-responder (MYD88 WT); • 3 Progressive Disease;

• 1 MDS/RAEB (Heavily pretreated; Pre-existing 5 q- in 10% metaphases; PR, progressed off treatment); • 1 Recurring thrombocytopenia (aggravated by hyper-splenism, resolved after splenectomy; SD on follow-up); • 1 Hematoma (post-procedure); • 1 Participant Decision (continues on follow-up);

32 Data Lock November 8, 2013

B

WHIM-like CXCR4 C-tail mutations in WM

Most common: CXCR4C1013G (S338X )

Warts, Hypogammaglobulinemia, Infection, and Myelokathexis.

Somatic WHIM-CXCR4 Mutations are present in WM patients: 8/30 (27%) by WGS ; 47/152 (31%) by Sanger Sequencing.

Hunter et al, ASH 2012; Blood 2013 33

34

Over 30 types of CXCR4 C-terminal somatic mutations in WM

Treon et al, Blood 2014

35

MYD88 and CXCR4 Mutation Status Impacts

Clinical Presentation of WM Patients

Treon et al, Blood 2014

MYD88WT

MYD88 WT L265P L265P L265P

CXCR4 WT WT FS NS

MYD88 WT L265P L265P L265P

CXCR4 WT WT FS NS

36

S338X somatic mutation impairs CXCR4 receptor

Internalization following SDF1a in transfected WM cells

Cao et al, ASH 2012, 2013

pBTK(Y223)

pERK44/42

pAkt(S473)

GAPDH

SDF1a

PCI-32765

AMD3100

Vector Wild type Mut S338X

CXCR4 WHIM Mutations lead to enhanced pERK and pAKT

activity in response to SDF1a.

Cao et al, ASH 2012; 2013

38

CXCR4

SDF-1a

WM CELL

39

SDF1a protects CXCR4S338X WM cells from

ibrutinib triggered apoptosis

Cao et al, ASH 2013

0.0

0.2

0.4

0.6

0.8

1.0

1.2

ANOVA p-values: Mutation Status = 1.88e-10 Therapy Cycle = 2.63e-36 Interaction effect = 8.93e-02

Serum IgM Mean Fold Change and Standard Error of the Mean by Cycle

Fo

ld C

han

ge R

ela

tive

to

Baselin

e

Baseline Cycle 2 Cycle 3 Cycle 6 Cycle 9 Cycle 12 Cycle 15 Cycle 18

CXCR4 WHIM-Like

CXCR4 Wild Type

Serial changes in serum IgM levels following Ibrutinib Stratified by CXCR4 status

0.8

1.0

1.2

1.4

1.6

1.8

ANOVA p-values: Mutation Status = 9.75e-07 Therapy Cycle = 8.13e-16 Interaction effect = 8.64e-02

Hb Mean Fold Change and Standard Error of the Mean by Cycle

Fo

ld C

han

ge R

ela

tive

to

Baselin

e

Baseline Cycle 2 Cycle 3 Cycle 6 Cycle 9 Cycle 12 Cycle 15 Cycle 18

CXCR4 WHIM-Like

CXCR4 Wild Type

Serial Hemoglobin levels following Ibrutinib Stratified by CXCR4 status

Data Lock November 8, 2013

p = 0.0065 Odds ratio = 0.115 95% CI 0.02-0.68

MYD88 N= VGPR/PR MR/SD/NR

L265P 48 31 (65%) 17 (35%)

Wild Type 5 2 (40%) 3 (60%)

CXCR4 N= VGPR/PR MR/SD/NR

WHIM 10 3 (30%) 7 (70%)

Wild Type 30 24 (80%) 6 (20%)

MYD88 /CXCR4 status and Ibrutinib Major Responses

p = 0.3536 Odds ratio = 2.68 95% CI 0.28-35.02

42

SDF1α + + - AMD3100 - -

- - - +

+ + +

+ + +

+ + +

- - -

- - -

- -

Cleaved PARP

Cleaved Caspase3

GAPDH

SDF1a protects CXCR4S338X WM cells from

chemotherapy triggered apoptosis

Cao et al, ASH 2013

Screening

Informed Consent and Registration

Ibrutinib

420 mg po daily

+ Plerixafor sq

qD

Progressive Disease or

Unacceptable Toxicity SD or Response

Continue x 26 cycles

Stop Ibrutinib/Pleixafor

Event Monitoring

Event Monitoring

44

Phase II Study of Ibrutinib plus Plerixafor in Relapsed/Refractory CXCR4WHIM WM Patients

MYD88

TLR4

TIR

AP

BTK MYD88

IκBα

TRAF6

NEMO

IKKα IKKβ

p50 p65 Nuclear Translocation and Signaling

IL1R

MYD88 L265P

Signal Pathway

Yang et al, Blood 2013

IBRUTINIB TAK1

46

IRAK1

remains

active in

WM

patients

on

ibrutinib

and

supports

survival of

WM cells.

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

Ibrutinib

Inh

ibit

ion

of

NF

kB

a b c d e f

IRAK1/4-In Ibrutinib +

IRAK1/4-In

g h i j k l

a + g b + h

c + i d + j

e + k f + l

IKBα-pS32

IKBα

GAPDH

Normalized Isobologram Combination Index Fraction Affected

Combination of Ibrutinib and IRAK inhibitors show synergistic

NFkB inhibition and WM cell killing (Yang et al, Blood 2013).

MYD88

TLR4

TIR

AP

BTK MYD88

IκBα

TRAF6

NEMO

IKKα IKKβ

p50 p65 Nuclear Translocation and Signaling

IL1R

TAK11

Targeting

MYD88 L265P

Signal Pathway

PI3Kδ

MYD88

ASO and

Homodimer

Inhibitors

Expression Ubiquitous Ubiquitous Leukocytes Leukocytes

EC50 (nM) >10,000 1419 2500 9

a b g d

Selective, oral inhibitor of PI3K-delta

Inhibits proliferation and induces apoptosis in

many

B-cell malignancies

Inhibits homing and retention of malignant B-cells

in lymphoid tissues reducing B-cell survival

Idelalisib

Class I PI3K Isoform

*Benson D et al. ASCO 2013, abstr 8526

Responses in relapsed/refractory indolent

NHL patients to idelalisib.

Gopal et al, NEJM 2014

Screening

Informed Consent and Registration

Idelalisib

150 mg

Twice a Day Progressive Disease or

Unacceptable Toxicity SD or Response

Continue x 26 cycles

Stop Ibrutinib/Pleixafor

Event Monitoring

Event Monitoring

51

Phase II Study of Idelalisib in Relapsed/Refractory WM Patients

A B

D

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Eff

ec

t

CAL101_0uM

CAL101_0.02uM

CAL101_0.06uM

CAL101_0.20uM

CAL101_0.63uM

CAL101_2.00uM

Ibrutinib Shows Synergistic WM Cell Killing with the

PI3K-delta Inhibitor Idelalisib (CAL 101)

Combination Index (CI)

< 1 : Synergism

Yang et al, ASH 2013 BCWM.1 Cells

What about MYD88 WT WM Patients?

• High levels of circulating B-cells.

• CD27+ disease more common (75-90%)

• Decreased IGHV3–23 rearrangements and

somatic hypermutation.

• Express mutations in the MLL2 gene.

• MLL2 encodes for the histone modifying

enzyme histone-lysine N-methyltransferase.

• MLL2 frequently mutated in follicular (89%)

and DLBCL lymphomas (32%).

Treon et al, NEJM 2012; Gachard et al, Leukemia 2013; Jiminez et al, Leukemia 2013.

54

Overall Survival in WM is Inferior with MYD88 Wild-Type Genotype

Treon et al, Blood 2014

Adjusted risk of death with MYD88WT

HR 10.54 (95% CI 2.40-46.2; p=0.002).

MYD88 L265P is present >90% of WM patients and triggers activation of Bruton’s Tyrosine Kinase (BTK) in WM cells.

The BTK inhibitor ibrutinib is associated with rapid reduction of serum IgM and improved HCT with an ORR of 83%, major RR of 65% in relapsed/refractory patients.

CXCR4 status impacts serum IgM and hemoglobin levels, and major response attainment.

Inhibitors to MYD88 and CXCR4 pathways represent novel approaches to the treatment of WM, alone and in combination.

55

Summary

Acknowledgements

Irene Ghobrial, MD

Jacob Laubach, MD

Sandra Kanan, NP

Jorge Castillo, MD

Gaurav Varma, MPH

Peter S. Bing, M.D. IWMF

Coyote Fund for WM

Waldenstrom’s Cancer Fund

NIH Spore Funding

Bailey Family Fund for WM

D’Amato Fund for Genomic Discovery

Edward and Linda Nelson Fund for WM

Bauman Family Trust

Tannenhauser Family Foundation

56

Diane Warren Sarah Daadi Rebecca Green Leslie Cheteyan Stephen Cropper Guang Yang, PhD Lian Xu, MA Yang Cao, MD

M. Lia Palomba, MD Ranjana Advani, MD Zach Hunter PhD IWMF

57

Bing Center for Waldenstrom’s Macroglobulinemia

www.bingcenterforwm.com

Chris Patterson (617) 632-6285

IWWM-8

London, UK

Aug. 12-16, 2014

www.wmworkshop.org

59

IN GRATITUDE TO THE IWMF FOR THEIR TIRELESS

EFFORTS ON BEHALF OF WM PATIENTS.

‘We shall defend our

island, whatever the

cost may be, we shall

fight on the beaches,

we shall fight on the

landing grounds, we

shall fight in the fields

and in the streets, we

shall fight in the hills;

WE SHALL NEVER

SURRENDER.”