- Diffuse large B-cell lymphoma (DLBCL) can be classified into molecular subtypes including germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes, as well as double hit lymphomas that have a poor prognosis. - New targeted agents in combination with R-CHOP have shown promising results for DLBCL subtypes, with proteasome inhibitors and immunomodulatory agents showing more activity in ABC DLBCL and BCL6 inhibitors, HDAC inhibitors, and etoposide showing potential in GCB DLBCL. - For double hit and MYC-positive lymphomas, intensive chemotherapy such as dose-adjusted

1. “ABC, GCB and Double-Hit diffuse large B
cell lymphoma: Does subtype make a
difference in Therapy Selection?”
Journal Club
Moderator: Dr. Suresh Babu MC
Presenter: Dr. Gita R Bhat

2. Authors: Grzegorz S. Nowakowski, Myron S. Czuczman
ASCO 2015, Educational Book

3. Overview
• Personalized therapy for treatment of patients with cancer is rapidly
approaching and is an achievable goal in the near future.
• DLBCL is the most common NHL
• 40% patients have refractory disease or disease that will relapse
after initial response
• 2 major biologically distinct molecular subtypes of DLBCL: GCB and
ABC
• Double hit lymphomas (approx 5%-10%) of patients and double
expressor lymphomas are aggressive and associated with poor
prognosis.
• Early clinical trials evaluating combination of novel targeted agents
in combination with R-CHOP have shown encouraging results.
• Hence, molecular classification: Prognostication + personalization of
therapy for DLBCL.

4. • Addition of rituximab (R) to CHOP in patients
with DLBCL: dramatic improvements in PFS
and OS.
• Inspite of this, 40% relapse or have refractory
disease.
• Various strategies to improve outcomes:
intensification of chemotherapy, use of
maintenance therapy, novel agents.

5. • Alternate regimens for front-line R-CHOP :
• Dose –dense R-CHOP 14: Phase III trial
showed no additional clinical benefit
• Dose-adjusted R-EPOCH
• R-CEOP 90
• Phase III trial: 1080 patients – No additional
clinical benefit was observed in patients
treated wih R-CHOP14 vs. R-CHOP21.

6. • Addition of novel agents (X) to R-CHOP:
• XR-CHOP
• “DLBCL has molecular heterogeneity”
• “X – targets specific oncogenic pathways”

8. • Classification of DLBCL based on the cell of
origin (COO):
• GCB (CD10, BCL6)
• Non-GCB
ABC (poor outcome)
Primary mediastinal B-cell types
• Associated with differences in clinical outcome

9. GCB DLBCL ABC DLBCL
Markers of germinal centre differentiation
(CD10 and BCL6)
-
- NF-κB pathway is constitutively active
High expression of NF-κB genes
BCL2 + BCL2+ (> 4 fold higher than in GCB DLBCL)
Arises from germinal centre B cell Post-germinal centre B cell, blocked
during plasmacytic differentiation
Outcome of GCB DLBCL and ABC DLBCL treated with RCHOP

10. Primary mediastinal B cell lymphoma
Arises from Thymic B cell
Predominantly in young women
Shares many features with CHL-NS
OCT-2 and BOB-1 (B cell transcription factors) are positive, Immunoglobulin
production is defective

11. Agents predominantly active in Non-GCB (ABC)
DLBCL
• Pathways that are constitutively activated in
ABC DLBCL:
• B-cell receptor (BCR) pathway
• Pathways downstream of BCR pathway
• Constitutive activation of NF-kB genes
• Proteasome inhibitors
• Immunomodulatory agents
• B-cell receptor signaling pathway inhibitors

12. B-cell receptor pathways
• Plays an important role in proliferation and
survival in B-NHL
• Targets:
• Spleen tyrosine kinase (SyK): Survival
• Bruton tyrosine kinase (BTK): BCR signaling
and maturation

13. Proteasome inhibitors
• Inhibit transcription factor NFkB
• Downstream pathway of BCR pathway
• Bortezomib combined with DA-EPOCH:
• ORR: GCB DLBCL (13%) vs. ABC DLBCL (83%)
• Median OS: GCB (10.8 months) vs. ABC ( 3.4
months)

14. IMiDs
• Structural and functional analogues of
thalidomide
• Regulate production of T-helper cells
• Inhibit cytokine production
• Inhibit production of TNFa
• Induce G0/G1 cell cycle arrest
• Inhibit angiogenesis through suppression of VEGF
and FGF
• Decrease NFkB activity

15. • Single agent Lenalidomide in relapsed
refractory/refractory NHL including DLBCL
• Phase II study, n= 217 patients
• DLBCL subpopulation: median PFS (2.7
months) and Response duration (4.6 months)

16. B-cell receptor signaling pathway
inhibitors - IBRUTINIB
• Bruton tyrosine kinase
inhibitor
• Forms covalent bond with
cysteine -481 in BTK
• High BTK specificity
• Daily oral dosing produces
24-hour BTK inhibition
• Blocks NF-kB activation in
ABC DLBCL
• Phase II study: ORR 22%, CR
5%, PFS: 1.6 months
(relapsed/ refractory DLBCL)

17. • SYK inhibitor: FOSTAMATANIB
• Phase II study, 23 patients
• Median PFS 2.7 months
• ORR: 22%

18. Agents with potential activity in GCB
DLBCL
• GCB DLBCL has better outcomes than ABC
subtype
• 20% of patients with GCB DLBCL relapse after
R-CHOP or R-CHOP like chemotherapy
• Associated with poor outcomes

19. • BCL6: highly expressed in GCB subtype
• Key transcription factor
• Translocations/ mutations enhance the
inhibitory effect of BCL6 on apoptotic stress
response
• This leads to tumor proliferation and
treatment failure

20. Therapeutic implications
A) Small molecule inhibitor of BCL-6:
• 79-6 complex
• Binds to the co-repressor binding groove of
the BCL6 domain and kills BCL-6 positive lines
B) HDAC inhibitors to overcome the effects of
BCL6 repression on p53

21. • C) Etoposide: Topoisomerase II inhibition –
ubiquitin mediated protein degradation and
transcriptional inhibition --- downregulates
BCL6
• DSHNHL study: better EFS in those who
received CHOEP vs. CHOP alone
• GCB DLBCL has higher incidence in younger
patients, hence they benefit more from the
addition of Etoposide.

22. • D) DA-EPOCH-R
• Inhibition of Topo II is optimised by
continuous delivery of drugs over 96 hours
• This ensures steady state concentration
• 5 years of follow-up: EFS (95% to 100%)
• E) EZH2: EZH2 inhibitors
• Gain of function mutations in EZH2 result in
increased H3K27 methylation

23. BCL2 inhibitors
• Members of BCL-2 family (BCL-2, BCL-XL, BCL-w, MCL-
1, BFL1/A-1, and BCL-B):
• Suppress apoptosis through interaction with, and
inactivation of, pro-apoptotic proteins such as BH3
• BCL2 inhibitors are active in ABC and GCB DLBCL
• In GCB: BCL2 is overexpressed as a result of
translocation
• In ABC: BCL2 is overexpressed at the protein level
• ABT-737 and ABT-263: target BCL-2, BCL-Xl and BCL-w
• ABT-199: potently and selectively inhibits BCL2

24. Front-line treatment: XR-CHOP
• Bor-RCHOP
• R2-CHOP
• IR-CHOP

25. Bor-RCHOP
• Untreated DLBCL or mantle cell lymphoma
• Ongoing Phase III RCT: CHOP vs. Bor-RCHOP in
DLBCL
ORR – 100%
CR or Cru: 86%
2-year PFS: 64%
2-year OS: 70%

26. R2-CHOP
• R2-CHOP:
• Lenalidomide-RCHOP
• Improves the poor prognosis usually reported
in non-GCB DLBCL
• Grade 3 and 4 AEs: Neutropenia (31%),
leucopenia (28%), thrombocytopenia (13%)

27. • Phase II trial: newly diagnosed DLBCL
• Treated with R2-CHOP vs R-CHOP
• Addition of Lenalidomide can improve the
poor prognosis in non-GCB population
2-year OS
GCB DLBCL Non-GCB
DLBCL
R2-CHOP 75% 83%
R-CHOP 78% 46%

28. • IR-CHOP:
• Phase I randomized trial, 33 patients
• Newly diagnosed DLBCL (22 patients) , Mantle
cell lymphoma, Follicular lymphoma
• ORR 100% (CR 64% and PR 36%)
• Most common AEs: Neutropenia, nausea,
thrombocytopenia, vomiting, anemia

29. • IR-CHOP:
• Bruton’s tyrosine kinase inhibitor
• Ibrutinib
• Phase Ib: Newly diagnosed DLBCL, mantle cell
lymphoma, follicular lymphoma

30. MYC-positive and Double-hit DLBCL
• MYC is a transcription factor
• Potent proto-oncogene
• Regulates 10%-15% of human genome
• Member of the helix-loop-helix leucine zipper
family of nuclear transcription factors
• Key to formation and maintenance of
germinal centres

31. • It can be activated via 3 modes: “Avalanche
effect”
• Translocation (5% - 14%)
• Copy gain (19% to 38%)
• Amplification (2%)
• Mutation (32%)

32. Target genes of MYC
Process involved Function Target genes induced Target genes repressed
Cell cycle Transit through cell cycle
G0 to S transition
Cyclin D2, CDK4 P21, p15, GADD45
Differentiation Blocks many cellular
systems
LDH, ribosomal proteins,
EIF4E, EI2A
Growth and metabolism Increase in cell size and
number
N-cadherin, integrin
Adhesion/migration Enables anchorage
dependent growth
Thrombospondin
Angiogenesis Induces angiogenesis IL 16, mir 17-92
Chromosomal instability Telomere aggregation,
ROS production
MAD2, TOP1, BUBR1,
Cyclin B1
Stem cell self renewal Potentiates induced
pluripotent stem cells
? ?
Transformation Drives tumorigenesis Several genes Several genes

33. MYC-associated pathways of
regulation of proliferation and survival

35. MYC-driven lymphomagenesis

37. Neoplasms with MYC gene
rearrangements
• Burkitt lymphoma
• t(8;14)
• Simple karyotype
• Sole chromosomal
abnormality
• DLBCL (7% - 14%)
• Unclassifiable B-cell
lymphoma (35%)
• Plasmablastic lymphoma
(50%)
• Plasma cell myeloma
(15% - 50%)
• Mantle cell lymphoma
• Complex karyotype
• Secondary genetic events

38. Why is this important?

39. • MYC rearrangement predicts an inferior outcome
in DLBCL
• These are seen in 58% - 83% of MYC-translocated
DLBCL
• OS when treated with RCHOP is ≤ 12 months
? Due to the MYC rearrangement itself
Double hit DLBCL
• Concurrent BCL2 translocation
• Less likely BCL6
Triple hit DLBCL
• Concurrent translocations in MYC+ BCL2 +BCL6

40. Amazing survival advantage!

41. Concurrent MYC + BCL2
MYC
1) Cell growth
2) Cell cycle transit
3) Angiogenesis
BCL2
1) Increased anti-
apoptosis
(drug resistance)

42. • Double-expressor lymphomas:
• High percentage of MYC and BCL2 protein
• By IHC staining
• Tumor cells should express at least 40% MYC
and at least 50% to 70% BCL2 positivity
• These are primarily ABC-like
• R-CHOP or CHOP-like chemotherapy: inferior
OS and PFS

43. FISH vs IHC for detecting Double hit DLBCL
• ? Effect of MYC alone (FISH or IHC) without
BCL2 on outcome
• BCL2+ MYC by IHC or FISH has worst outlook
“Assessment of MYC and BCL2 expression by
IHC represents a robust, rapid, and
inexpensive approach to risk-stratify patients
with DLBCL at diagnosis”

44. • DHL patients have several poor prognostic
factors:
• Median age: 7th decade
• Stage III/IV disease
• IPI: High intermediate/high
• Elevated LDH
• High frequency of extra-nodal sites (excluding
CNS)

45. • Of the several intensive chemotherapy
regimens used:
• R-EPOCH
• (1) has curative potential in BL
• (2) is better tolerated than most dose-
intensive regimen
• (3) appears to have similar efficacy compared
to other dose-intensive therapies

46. Combination chemotherapy in Double-hit lymphoma

47. Initial therapy for Double hit lymphoma
• Petrich AM et al. Blood 2014; 124:2354-2361
• Retrospective study of outcomes in 23 US centres
over 12 years:
• 311 patients with newly diagnosed DLBCL (154),
BCLU (150), FL (7)
• MYC-R and BCL2 (87%) or BCL6 (5%) by
FISH/cytogenetics
• 76% raised lDH, 33% ≥3* ULN
• 65% stage IV, 41% BM +ve, &% CNS +ve

49. Comparison of long-term, progression-free, and overall survival.
Adam M. Petrich et al. Blood 2014;124:2354-2361
©2014 by American Society of Hematology

50. Role of stem cell therapy
• Autologous SCT in those who achieve CR:
• Does not significantly change clinical
outcomes
• Inherent rapid tumor cell growth and inherent
drug resistant DHL cells (Minimal residual
disease)

51. • Allogenic SCT is unlikely to have a major effect
since:
• 1) limited data from a small number of
selected patients
• 2) the risk of relapsed disease while awaiting
graft-vs-lymphoma to occur
• 3) the need for a suitable HLA-compatible
donor
• 4) chronic GvHD

52. Overall survival by SCT versus observation in first complete remission.
Adam M. Petrich et al. Blood 2014;124:2354-2361
©2014 by American Society of Hematology

53. • Adverse factors for OS at diagnosis:
• Leukocytosis
• LDH >3*ULN
• Advanced Ann Arbor stage
• CNS involvement

54. • Hence,
• DA-R-EPOCH induction + CNS prophylaxis is a
reasonable approach
• Further escalation of chemotherapy, especially
in the salvage setting is unlikely to be of
benefit…. Hence, novel agents…

55. Waiting in the wings….

56. ABT - 199 Platelet sparing BCL2 inhibitor (BH3 mimetic) restores
apoptosis
Bromodomain inhibitors Down-regulation of MYC-associated transcription:
Decreased cell proliferation and inhibition of MYC-driven
neoplasms
CAR-T cells Autologous T-cell mediated killing of CD19-positive
lymphoid neoplasm
Aurora kinase inhibitors
(Alisertib)
Aurora kinase is required for tumor maintenance of MYC-
driven lymphoma
mTor inhibition mTor plays an important role in tumor maintenance by
MYC in B lymphocytes
Second generation
Proteasome inhibitor
(Ixazomib)
Degrade MYC and can induce lymphoma cell death
PI3K inhibition In GCB-DLBCL: Loss of PTEN leads to activation of
PI3K/AKT pathway ___ MYC upregulation
Inhibition of
mitochondrial peptide
deformylase
Apoptosis in MYC-over expressing hematopoietic
neoplasms
SIRT4 protein Suppresses tumor formation in MYC-induced B-cell
lymphoma

57. Key points
• ABC subset of DLBCL is biologically distinct
• Associated with poor outcomes when treated with a standard therapy.
• Activation of the clonic B-cell receptor pathway allows for therapeutic
targeting.
• Targeted agents in relapsed DLBCL can be combined with R-CHOP in front-
line therapy of DLBCL.
• The germinal center B-cell (GCB) subset of DLBCL is associated with better
outcomes and may require different therapeutic approaches.
• Double-hit lymphoma (DHL) is responsible for a substantial number of
relapses in GCB DLBCL
• All newly diagnosed DLBCL biopsy samples should be tested for DHL by
fluorescent in situ hybridization and by immunohistochemistry for double-
expressor DLBCL
• Whenever possible, patients should be referred to participation in clinical
trials
• DHL: DA-R-EPOCH plus central nervous system prophylaxis until more
effective novel targeted agents for this lymphoma subtype are developed

58. Thank you!