1. Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL) with Non-Germinal center B-cell Phenotype is associated with a Higher response to Lenalidomide (L) or in Combination with
Rituximab(R)
Francisco Hernandez-Ilizaliturri MD Farhana Malik MBBS Myron Czuczman MD
Departments of Medical Oncology, Immunology and Pharmacology
Roswell Park Cancer Institute

2. Classification of Newly Diagnosed Diffuse Large B-Cell Lymphoma (DLBCL) According to the Hans Criteria Defines Two Groups of Patients with Different Clinical Outcomes Following Systemic Rituximab-Multi Agent Anthracycline-Based Therapy

3. Evolution in the management of aggressive B-cell lymphomas
Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma diagnosed in the Western Hemisphere
Patients with DLBCL exhibit a heterogeneous clinical behavior and prognosis
Several biomarkers that predict the clinical outcome of DLBCL patients had been identified and validated (i.e. Bcl-2 expression, IPI score or gene profiling studies)
The addition of rituximab to systemic chemotherapy has improved the clinical outcome of DLBCL patients, challenging previously established biomarkers of response

4. Rosenwald et Al, NEJM 2002; 346:1937-47
Distinct types of DLBCL identified by gene expression profiling
Rosenwald et Al, NEJM 2002; 346:
Studies included patients with DLBCL treated with CHOP or CHOP-like chemotherapy prior to rituximab
Alizadeth A, et al. Nature 2000; 403:

5. Algorithm for Subtype classification of DLBCL
CD10
GCB
Bcl-6
MUM1
Non-GCB + -
The concordance rate of the Han’s Algorithm to gene profiling studies is 80%
Hans et al, Blood 2004, 103:

6. Differences in outcomes among patients with DLBCL treated with R+CHOP subdivided by IHC

7. To study clinical differences between GCB- and non-GCB DLBCL treated at RPCI
Retrospective study of 191 patients DLBCL treated at our Institution between 2000 and 2007 with the following characteristics
Previously untreated DLBCL
Patients that received front line therapy with rituximab in combination with CHOP or DA-EPOCH
Tumor specimen available for immunohistochemistry (IHC) studies
CD20, CD10, BCL2, BCL6 and MUM1
Patients were excluded from the analysis if any of the following:
Incomplete clinical data
Transformed lymphoma and primary CNS DLBCL
HIV related DLBCL
Demographic, clinical and pharmacological (i.e. cumulative doses of rituximab, chemotherapy and growth factor support) were included in the analysis

8. Patients were classified as GCB- or non-GCB DLBCL using the Hans algorithm (Hans et al, Blood 2004, 103: )
Differences in end-point studies were evaluated between GCB- and non-GCB patients
Repsonse rate
Cumulative chemoimmunotherapy doses received
Overall survival (OS)
Progression free survival (PFS)

11. DLBCL with a non-GCB phenotype by IHC had an inferior PFS following R-CHOP-21 than GCB-DLBCL
Log Rank P = 0.017
GCB DLBCL
Non-GCB DLBCL
The median survival for non-GCB DLBCL was 45.1 months, whereas the median survival for patients with GCB-DLBCL has not be reach.

12. DLBCL with a non-GCB phenotype by IHC had an inferior overall survival following R-CHOP-21 than GCB-DLBCL
Non-GCB DLBCL
GCB DLBCL
P = 0.037
The median survival for non-GCB DLBCL was 75.4 months, whereas the median survival for patients with GCB-DLBCL has not be reach.

13. In summary
Our data suggest that the Hans algorithm can predict clinical outcomes of patients with DLBCL undergoing frontline chemo-immunotherapy
Patients with Non-GCB DLBCL while having a comparable initial overall response rate (CR and PR) to R+CHOP had a shorter PFS and OS than GCB-DLBCL
Non-GCB DLBCL represent a subgroup of DLBCL for which innovative therapeutic strategies targeting key regulatory pathways in the induction and/or maintenance setting are need in an attempt to improve their PFS and OS

14. De Novo CD20+ DLBCL stage II,III or IV Tumor Biopsy for GEP studies
CALGB Phase III Randomized Study of R-CHOP vs. Dose-Adjusted EPOCH-R with Molecular Profiling in Untreated De Novo Diffuse Large B-Cell Lymphomas
R+CHOP x 6
De Novo CD20+ DLBCL stage II,III or IV
Tumor Biopsy for GEP studies
Randomization
R-DA-EPOCH x 6

15. Lenalidomide in diffuse large B-cell lymphoma (DLBCL): correlating response with tumor characteristcs

16. Thalidomide analogues: immune-mediated inflammatory disease (IMiDs) in B-cell lymphomas
Thalidomide/IMiDs target MM cells in the bone marrow (BM) microenviroment O O H N
C. Thalidomide/IMiD N O
MM cells O
IL-6  TNFa  IL-1b 
B. Thalidomide/IMiD
Thalidomide
BM stromal cells O O
A. Thalidomide/IMiD H
ICAM-1 N N O
BM vessels N H 2
VEGF  bFGF 
CC-5013 (Revimid)
IL-2  IFN 
PBMC O O H
E. Thalidomide/IMiD
D. Thalidomide/IMiD N
CD8+ T cells NK cells N O
MM = multiple myeloma; VEGF = vascular endothelial growth factor PBMC = peripheral-blood mononuclear cells; NK = natural killer TNF = tumour necrosis factor O N N N H H H 2 2 2
CC-4047 (Actimid)
Davies FE, et al. Blood, 2001;98:210–6 Hayashi T, et al. Blood 2002;100:314b (Abstract 4800)

17. NF-kB target genes are highly expressed in activated B cell-like diffuse large B-cell lymphoma
Courtesy of L. Staudt

18. IMiDs in vitro decrease NFB activity and arrest DNA synthesis in NHL cells
Raji cells
CC5013
40,000
30,000
20,000
10,000
400,000
300,000
200,000
100,000
CPU 48 hours Raji cells
Relative light units
µg/mL
CC4047
400,000
300,000
200,000
100,000
CPU 48 hours Raji cells
Placebo
Negative control
Positive control
CC4047
CC5013
µg/mL
Hernandez-Ilizaliturri FJ, et al. Clin Can Res 2005;11:5984–92

19. Stromal-1 signature encodes extracellular matrix components and macrophage/myeloid restricted proteins (favourable)

20. The stromal-2 signature encodes regulators and components of angiogenesis (unfavourable)

21. Tumor micro vessel density decreases after treatment with lenalidomide
Placebo CD31 staining
Lenalidomide CD31 staining
Raji xenografts
Actimid CD31 staining
Bars show means Error bars show mean ± 1.0 SE
125
100 75 50 25 *† †
MVD (vessels/llpf) *
Placebo
Lenalidomide
Actimid
*p=0.009 †p=0.005
Reddy N, et. al. Br J Haematol 2007, 140:36-45

22. Lenalidomide induces CRs and PRs in patients with relapsed and treatment-refractory CLL
Plasma levels of TNF-, soluble TNF receptor 1 (TNF-R1), interferon (IFN)-, VEGF, bFGF, interleukin (IL)-1β, -2, -6, -8, -10, -12, IL-1 receptor antagonist (IL-1RA), and soluble IL-2 receptor (IL-2R) were measured at different time intervals pre and post-treatment
A significant reduction in plasma VEGF level was observed on day 28 in four patients that achieved a response (mean reduction of 55.6pg/mL [± 15.3], p=0.036) and on day 90 in four patients with SD or clinical improvement (mean reduction of 50.9pg/mL [± 3.3], p=0.003).
Ferrajoli A, et al. Blood 2006;108:94a (Abstract 305)

23. Cytokine secretion by DC after in-vitro exposure to lenalidomide* 20 15 10 5
INF- *p=0.014
†p=0.004 †
pg/mL *
4,000
3,000
2,000
1,000
MCP-1 *p<0.001
†p=0.001 † *†
DMSO Lenalidomide Actimid
pg/mL
700
600
500
400
300
200
100
TNF-a *p=0.013
†p=0.024 † *† *
DMSO Lenalidomide Actimid
pg/mL *†
DMSO Lenalidomide Actimid
Reddy N, et. al. Br J Haematol 2007, 140:36-45

24. Differences in the number and pattern of NK cells infiltrating the tumour bed of lymphoma-bearing SCID mice treated with IMiDs
Placebo-treated infiltration of NK-cells (CD49b+) in the periphery of the tumour
Raji xenografts
Bars show means Error bars show mean ± 1.0 SE 60 40 20 * † *†
Lenalidomide-treated infiltration of NK-cells (CD49b+) within the tumour
Tumour infiltrated with NK-cells (%)
Placebo
Lenalidomide
Actimid
*p=0.012 †p=0.015
Reddy N, et. al. Br J Haematol 2007, 140:36-45

25. IMiD enhancement of rituximab-dependent ADCC ex vivo is mediated via co-stimulation of NK-cells by DCs
Co-stimulation with DCs
Without co-stimulation with DCs 30 20 10 30 20 10
Raji
Raji
DMSO
Lenalidomide
Pomalidomide
Bars show means
DMSO
Lenalidomide
Pomalidomide
Bars show means
p<0.007
Specific lysis (%)
Error bars show mean ± 1.0 SE
Specific lysis (%)
Error bars show mean ± 1.0 SE
Rituximab Isotype Splenocytes
Rituximab Isotype PBMC
Reddy N, et. al. Br J Haematol 2007, 140:36-45

26. Median survival (days)
Pomalidomide in combination with rituximab improves survival in lymphoma-bearing severe combined immunodeficiency (SCID) mice
1.2
1.0
0.8
0.6
0.4
0.2
Mice/group (n=15)
Median survival (days)
95% CI
Placebo 21
20–22
Rituximab 38
26–50
Pomalidomide
21–21
Pomalidomide + rituximab 74
70–78
Pomalidomide* + rituximab
Cumulative survival
Pomalidomide
Rituximab*
Placebo
Time to development of limb paralysis (days)
*p=0.0012
CI = confidence interval
Hernandez-Ilizaliturri FJ, et al. Clin Can Res 2005;11:5984–92

27. Median survival (days)
Lenalidomide in combination with rituximab improves survival in lymphoma-bearing SCID mice
1.2
1.0
0.8
0.6
0.4
0.2
Mice/group (n=15)
Median survival (days)
95% CI
Placebo 21
18–24
Rituximab 45
32–58
Lenalidomide
20–22
Lenalidomide + rituximab 58
43–73
Lenalidomide* + rituximab
Cumulative survival
Lenalidomide
Rituximab*
Placebo
Time to development of limb paralysis (days)
*p=0.167
Hernandez-Ilizaliturri FJ, et al. Clin Can Res 2005;11:5984–92

28. Lenalidomide Monotherapy in Relapsed or Refractory Aggressive Non-Hodgkin’s Lymphoma: NHL-002
Patients self-administered oral lenalidomide (25 mg once daily) on days 1 to 21 of every 28-day cycle. Patients continued therapy for 52 weeks as tolerated or until disease progression.
Histology
No. of Patients CR
CRu PR SD PD
ORR (%)
Aggressive NHL 49 2 4 11 21 35
Diffuse large B-cell lymphoma 26 1 7 14 19
Follicular center lymphoma, grade 3 5 60
Mantle-cell lymphoma 15 6 53
Transformed low-grade lymphoma 3 33 O O O H N N O
Wiernik P, et al. Journal of Clinical Oncology, 26; 2008: N H 2

29. Lenalidomide Monotherapy in Relapsed or Refractory Aggressive Non-Hodgkin’s Lymphoma
The estimated median duration of response was 6.2 months (range, 0 to 12.8 months), and median PFS was 4.0 months (range, 0 to 14.5 months).
Wiernik P, et al. Journal of Clinical Oncology, 26; 2008:

30. Confirmation of the Efficacy and Safety of Lenalidomide Oral Monotherapy in Patients with Relapsed or Refractory Diffuse Large-B-Cell Lymphoma: Results of An International Study (NHL-003)
NHL-003 evaluated the efficacy of single-agent lenalidomide in patients with relapsed/refractory aggressive NHL that had received at least one prior treatment and had measurable disease (Median 3, range 1-6)
Lenalidomide was administered at 25mg/day p.o. on days 1–21 of a 4-week cycle until disease progression or unacceptable toxicity
Results in patients with DLBCL (N=73) demonstrated:
The overall response rate to lenalidomide was 29% (21/73)
While some complete response were observed (4%, 3/73), most of the patients achieved a partial remission (25%, 18/73).
Eleven patients (15%) had stable disease
The most common grade 3 or 4 adverse events were neutropenia (32%), thrombocytopenia (15%), asthenia (8%) and anemia (7%).
Czuczman M, et al. Blood (ASH Annual Meeting Abstracts) : 268a

31. Algorithm for Subtype Differentiation
CD10
GCB
Bcl-6
MUM1
Non-GCB + -
Iqbal J et al. Leukemia 2007, 1–12
Hans et al, Blood 2004, 103:

32. Can we predict clinical response to Lenalidomide in DLBCL patients?
Retrospective study of patients with DLBCL treated with lenalidomide at Roswell Park Cancer Institute (RPCI)
Patients were divided into two cohorts using the criteria proposed by Hans et al and Igbal et al :
Germinal center B-cell-like (GCB)
Non-GCB
Tumor biopsies are routinely stained for MUM1, CD10, Bcl-6 and Ki67 by the Pathology Department at RPCI
Responses to lenalidomide were assessed by standard and/or revised Cheson criteria
Differences in response rate, duration of response to lenalidomide and overall survival were analyzed using the software program SPSS 14

33. Can we predict clinical response to Lenalidomide in DLBCL patients?
Sample size 19, 11F/8M
Histological Diagnosis:
DLBCL = 13
FL and DLBCL (Composite) = 5
Transformed NHL = 1
IHC classification of the patients:
Non-GCB = 9
GBC = 9
Unknown = 1 (Bcl-6 positive, T-cell rich DLBCL)
Median Age 64 (43 to 89)
Median number of prior therapies = 4 (2 – 13)
Median cycles of lenalidomide = 3 (1 – 20)

34. Differences in response rate between Non-GCB and GCB-like DLBCL to lenalidomide*
Median number of prior treatment: Non-GCB = 4.88 (+/- 1.03) vs. GCB = 4.33 (+/- 0.64), Chi square P = 0.46
No differences in IPI score, histology, stage or other demographic characteristics were seen at time of lenalidomide Rx between the two groups

35. Response to lenalidomide single agents in patients with DLBCL
All Patients (n = 18)
GCB-phenotype (N=9)
Non GCB-phenotype (N=9)
Median number of lenalidomide cycles
3.5 (1-24)
2 (1-21)
7 (1-24)
Responses as per revised Cheson criteria (PET scan included):
Complete response (CR)
Partial response (PR)
Stable disease (SD)
Progression of disease (PD)
Overall response (CR + PR)
5 (27.7%)
3 (16.6%)
1 (5.5%)
9 (50%)
8 (44.4%)
1 (11.1%)
0 (0%)
7 (77.7%)
1 (11.1%)*
4 (44.4%)
3 (33.3%)
2 (22.2%)
7 (77.7%)* P=0.011
Responses as per 1999 Cheson criteria (PET scan excluded):
4 (22.2%)
2 (11.1%)
7 (38.8%)
3 (44.4%)
6 (66.6%)*P=0.048
Deaths (number, %)
5 (55.5%)
PFS (median, months)
4.9 (1.4 to 8.6)
2.4 ( )*
11.2 (3.9 to 18.5)* *P=0.008
DLBCL = diffuse large B-cell lymphoma, PET = positron electron tomography

36. Progression free survival following Lenalidomide therapy in DLBCL according to histological subtype
DLBCL Type
Mean (Days)
Estimate
Std. Error
95% Confidence Interval
Lower Bound
Upper Bound
Non-GCB
82.838
GCB
62.125
9.124
Overall
63.811

37. Progression free survival following Lenalidomide therapy in pure DLBCL
DLBCL Subtype
Mean
Estimate
Std. Error
95% Confidence Interval
Lower Bound
Upper Bound
Non-GCB
96.152
GCB
.000
Overall
83.328

38. DLBCL with a non-GCB phenotype by IHC had a better overall survival following lenalidomide

39. Response rate in DLBCL with a non-GCB phenotype by IHC treated with rituximab and lenalidomide
Patients with refractory/relapsed NHL treated at University of Bologna with rituximab in combination with lenalidomide (mean number of cycles =4)
Tissue array submitted to RPCI for MUM-1, Bcl-6, and CD10 IHC
Histological Diagnosis (revised and re-classified at RPCI):
DLBCL = 10
FL3a/b = 3
Transformed NHL = 1
IHC classification of the patients by the Hans algorithm was feasible in 10pts:
Non-GCB = 8
GBC = 0
Other (FL) = 2
ORR for all cases submitted = 50% (7/14)
ORR for Non-GCB = 50% (4/8)

40. In summary
Lenalidomide monotherapy or in combination with rituximab are active salvage therapies in relapsed/refractory DLBCL
Our data strongly suggest that two previously identified groups of patients with DLBCL (GCB vs. non-GCB) appear to have dramatically different degrees of responsiveness to lenalidomide with or without rituximab in the relapsed/refractory setting
Tumor specimens from patients treated at two additional institutions (i.e. Mayo Clinic and the John Theurer Cancer Center at Hackensack University Medical Center) are currently being evaluated and classified in order to further validate our findings

41. Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma
Gene expression profiling (GEP)
Immuno-histochemistry (IHC)
Relapsed/Refractory DLBCL (N=49)
CD10
Bcl-6
MUM1
Biopsy
Proceed to Part B if clinically indicated
Part A Bortezomib (N=23)
ABC DLBCL (N=5)
GCB DLBCL (N=10)
ABC DLBCL (N=12)
GCB DLBCL (N=12)
Treat until disease progression or maximum allowable cycles
DLBCL subtype classification by GEP or IHC
Part B Bortezomib + DA-EPOCH (N=44)
ABC DLBCL (N=12)
GCB DLBCL (N=15)
Treat until disease progression or maximum allowable cycles
Dunleavy et al. Blood. 2009; 113:

42. Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma
P=0.0026
Dunleavy et al. Blood. 2009; 113:

43. Relapse Refractory DLBCL
Overall study schema for proposed Phase III registrational trial of lenalidomide in r/r DLBCL
Lenalidomide
N=25
Lenalidomide
N=74
Non-GCB
Non-GCB
Control
N=25
Control
N=74
Relapse Refractory DLBCL
Stratify by
IHC subtyping
Lenalidomide
N=25
Lenalidomide
N=74
GCB
GCB
Control
N=25
Control
N=74
Stage 1
N = 100
Stage 2
N = 148 or 296
Go/No-go Analysis

44. Lymphoma Translational Research Group Lenalidomide project
Medical Oncology/immunology
Myron S. Czuczman M.D.
Francisco J. Hernandez-Ilizaliturri M.D.
Asher Chanan-Khan M.D.
Zale Berstein M.D.
Jeyanthi Ramanarayanan M.D.
Philip McCarthy M.D.
Kelvin Lee M.D., Ph.D.
Navine Bangia Ph.D.
Elizabeth Repasky Ph.D.
Departments of Hematology, Anatomic Pathology, and Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN
Thomas Witzig MD
William Macon MD
Department of Hematology and Pathology, Bologna University School of Medicine, Bologna, Italy,
Pier Luigi Zinzani MD
Stefano A. Pileri MD
The John Theurer Cancer Center at Hackensack University Medical Center, Hackensack NJ.
Andrew Goy MD
Pathology/molecular diagnostics
Maurice Barcos M.D.
George Deeb M.D.
Paul Wallace Ph.D.
Peter Sterostick Ph.D.
Ann Marie Block Ph.D.
IMIDs pre-clinical project
Malik Farhana MBBS
Nishitha Reddy M.D.
Beata Holkova M.D.