1. Prognostic Markers in B-cell Lymphomas
Sergei Syrbu MD, PhD
Department of Pathology
Hematopathology/Immunopathology

2. Disclosure
I have no actual or potential conflicts of interest in relation to the content of this presentation. I will not be discussing off-label use of drugs or devices.

3. Prognostic Markers in B cell lymphomas
DLBCL
Clinical - International Prognostic Index (IPI) and Revised-IPI
Gene expression profiling
- Affymetrix U133 Plus on FT
- Gene expression in FFPE tissue (NanoString Lymph2CX)
- IHC algorithms for GCB and ABC phenotypes
Double hit lymphoma (DHL) and atypical “DHL”
IHC “DHS” (DPL) or MYC and BCL-2 double positive lymphoma
CD5+
Absolut Monocyte Count (AMC) and Absolute Lymphocyte Count (ALC) Prognostic Index (AMLCPI)
Tumor associated macrophage polarization in DLBCL and Hodgkin lymphoma
Serum free light chains
BCL6 expression
P53+++/P21-
P21+
BCL2+
MCL - Ki67, P53, SOX11
FL - Serum cytokines and chemokines

4. Diffuse large B cell lymphoma
Highly heterogeneous (clinically, immuno- phenotypically, morphologically, biologically) group of diseases
Most common lymphoma in North America (30-40%)
24,000 new cases every year
Affects all ages and genders
~40% of patients with DLBCL suffer relapses and die of disease

5. Prognostic Markers in DLBCL
Clinical - International Prognostic Index (IPI)
- Age >60
- Stage III or IV
- Elevated LDH
- Performance status 2 or higher
- Two or more extranodal sites
Depending on IPI score the 5-year overall survival ranges from 26% to 73%.
IPI is also age-adjusted (<60 and >70)

6. OS of DLBCL patients Data are from patients with TMA available
according to the IPI for the 3 patient cohorts:
r-CHOP 347 patients (panel 1a), c-CHOP
289 patients (panel 1b) and e-CHOP
878 patients (panel 1c).The log-rank P value
was < for each cohort.
OS of DLBCL patients. Data are from patients with TMA available according to the IPI for the 3 patient cohorts: r-CHOP 347 patients (panel 1a), c-CHOP 289 patients (panel 1b, control for second R-CHOP arm) and e-CHOP 878 patients (panel 1c, early CHOP before R-CHOP era). The log-rank P value was < for each cohort.
Gilles Salles et al. Blood 2011;117:
©2011 by American Society of Hematology

7. IPI
One of the most clinical predictor of survival in DLBCL and can identify patients as Low (0,1), Low-Intermediate (2), High-Intermediate (3) and High Risk (4,5)
Since Rituximab was introduce a revised IPI (R-IPI) was proposed, which stratifies patients into 3 groups – Very Good (0), Good (1,2) and Poor (3,4,5)
The IPI does not capturer the biological spectrum of DLBCL!

8. Cell-of-origin - COO DLBCL Affymetrix on FT
Figure 4 Relationship of DLBCL subgroups to normal B-lymphocyte differentiation and
activation. The data in the left panel are taken from Fig. 3c. The right panel depicts gene
expression data from the following normal B-cell samples: (1) Total CD19+ blood B cells;
(2) Naive CD27- blood B cells; (3) Memory CD27+ blood B cells; (4) cord blood CD19+ B
cells; (5) blood B cells; anti-IgM 6 h; (6) blood B cells; anti-IgM + IL-4 6 h; (7) blood B cells;
anti-IgM + CD40 ligand 6 h; (8) blood B cells; anti-IgM + CD40 ligand + IL-4 6 h; (9) blood
B cells; anti-IgM 24 h; (10) blood B cells; anti-IgM + IL-4 24 h; (11) blood B cells; anti-IgM
+ CD40 ligand 24 h; (12) blood B cells; anti-IgM + CD40 ligand + IL-4 24 h; (13) blood B
cells; anti-IgM + CD40 ligand (low concentration) 48 h; (14) blood B cells; anti-IgM +
CD40 ligand (high concentration) 48 h; (15) tonsil germinal centre B cells; (16) tonsil
germinal centre centroblasts. See Supplementary Information for full data.
Affymetrix on FT

9. Determining cell-of-origin subtypes of DLBCL using gene expression in FFPE tissue (Lymph2Cx)
NanoString technology was used to determine expression of 20 genes (15 target genes and 5 housekeeping) in FFPET-derived RNA
>95% concordance of COO assignment by GEP
7% were designated as “unclassified”
Turnaround time – 36 hrs
Scot et al. Blood :

10. Lymph2Cx on FFPET Affymetrix U133 Plus on FT
Patient outcomes according to COO in the independent validation cohort.
Lymph2Cx on
FFPET
Affymetrix U133
Plus on FT
Patient outcomes according to COO in the independent validation cohort. (A) Progression-free survival in the COO groups as determined by the Lymph2Cx assay. (B) Overall survival in the COO groups as determined by the Lymph2Cx assay. (C) Progression-free survival in the COO groups determined by the gold standard method applying the previously described model6 to gene expression on FT. (D) Overall survival in the COO groups determined by the gold standard method. The P values are from log-rank tests comparing the ABC and GCB groups. The log-rank tests are 1 sided in the direction of greater hazard for ABC. RR, relative risk (with the 95% confidence interval in brackets) associated with the ABC group compared with the GCB group. The groupings in A and B are from the results at the Molecular Characterization Laboratory (Frederick National Laboratory for Cancer Research). Results from the Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, BC Canada, are shown in supplemental Figure 4.
David W. Scott et al. Blood 2014;123:
©2014 by American Society of Hematology

11. Determining cell-of-origin subtypes of DLBCL using gene expression in FFPE tissue (Lymph2Cx)
NanoString technology was used to determine expression of 20 genes (15 target genes and 5 housekeeping) in FFPET-derived RNA
>95% concordance of COO assignment by GEP
7% were designated as “unclassified”
Turnaround time – 36 hrs
Scot et al. Blood :

12. IHC Methods for Predicting Cell of Origin and Survival in Patients With DLBCL Treated With Rituximab
This study compares some of those algorithms and also proposes some modifications
Paul N. Meyer et al. JCO 2011;29:

13. Immunohistochemical algorithms examined in this study.
Immunohistochemical algorithms examined in this study. The cutoff for positivity is 30% of the tumor cells showing expression unless otherwise indicated (Choi and Muris algorithms). In the Tally algorithm, antibody results are not examined in a particular order. Two antigens of germinal center B cells and two antigens of activated B cells are examined. The immunophenotype with more positive antigens is determined. If an equal number of germinal center B-cell immunophenotype (GCB) and activated B-cell immunophenotype (ABC) antigens are positive, then LMO2 determines the phenotype. Choi*, modified Choi algorithm; Hans*, modified Hans algorithm.
Meyer P N et al. JCO 2011;29:
©2011 by American Society of Clinical Oncology

14. Overall survival of patients with diffuse large B-cell lymphoma according to immunophenotype by each algorithm.
Overall survival of patients with diffuse large B-cell lymphoma according to immunophenotype by each algorithm. The number of patients (n) is listed next to the immunophenotype result, and n varies among the algorithms because of loss of tissue cores during staining. The probability that the two patient groups have equivalent survivals and the algorithm used is noted in the lower left corner of each graph. GCB, germinal center B-cell immunophenotype; ABC, activated B-cell immunophenotype.
Paul N. Meyer et al. JCO 2011;29:
©2011 by American Society of Clinical Oncology

15. Event-free survival of patients with diffuse large B-cell lymphoma according to immunophenotype by each algorithm.
Event-free survival of patients with diffuse large B-cell lymphoma according to immunophenotype by each algorithm. The number of patients (n) is listed next to the immunophenotype result, and n varies among the algorithms as a result of loss of tissue cores during staining. The probability that the two patient groups have equivalent survivals and the algorithm used is noted in the lower left corner of each graph. GCB, germinal center B-cell immunophenotype; ABC, activated B-cell immunophenotype.
Paul N. Meyer et al. JCO 2011;29:
©2011 by American Society of Clinical Oncology

16. Conclusion
The Hans and Choi algorithms are useful to determine the cell of origin of DLBCL and can separate patients into prognostic groups, with or without the use of BCL6.
A new Tally algorithm showed the greatest concordance with microarray results .
Although the Tally and Choi algorithms may be preferred, the pathologists should be allowed to choose the most appropriate algorithm for their practice.
The immunohistochemical algorithms are sufficiently robust to allow cell of origin determination for future therapies based on cell of origin.

17. Like the IPI, COO subtyping doesn’t identify patients with highly aggressive DLBCL since these patients will be in both subgroups!

18. R-CHOP is inadequate in many DLBCL subsets and high-risk groups
Freq
PFS OS
ABC DLBCL
30-50%
2-yr 28%
2-yr 46%
DHL
5-7%
1-yr 33%
<1 yr
DPL (MYC+BCL2)
34%
5-yr 27%
5-yr 30%
Elderly DLBCL >60 yr
50%
5-yr 50%
5-yr 58%
High IPI
45%
4-yr 53%
4-yr 55%
NCI CTPM: Recommendations of the DLBCL Subcommittee
November 21, 214
Sonali Smith, Kristie Blum, David Maloney, Greg Nowakowski,
Laurie Sehn, Michael Williams, Wyndham Wilson

19. Double hit lymphoma (DHL) and atypical DHL
DHL - defined as an aggressive high-grade B-cell lymphoma with translocations involving both MYC and BCL2 or BCL6, BCL3, or CCND1 (MYC/BCL2 most common)
Atypical DHL - abnormalities of MYC and BCL2 other than coexistent translocations:
1. MYC translocation + extra BCL2 copies
2. t(14;18) + extra copies of MYC
3. Extra copies of MYC and BCL2
Represents 6–14% of patients

20. Double hit lymphoma (DHL) and atypical DHL (aDHL)
CNS and bone marrow involvement are common
Most cases of DHL and aDHL have morphologic features of DLBCL or B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma
The majority are of GCB-cell immunophenotype with a high proliferation rate
There is no established standard therapy for DHL patients - median OS of <2 years
Characterized by advanced stage disease, extranodal involvement, and high serum LDH levels.

21. Comparison of pathologic features of atypical and typical MYC/BCL2 DHL
Atypical MYC/BCL2 DHL
MYC/BCL2 DHL
 DLBCL
75% (30/40)
47% (36/76)
 BCLU
18% (7/40)
46% (35/76)
 Other
8% (3/40)
7% (5/76)
Immunophenotype
 CD10*
72% (28/39)
99% (71/72)
 BCL6
93% (25/27)
95% (36/38)
 BCL2 (>50%)
94% (33/35)
90% (61/68)
 MYC (>40%)
70% (14/20)
75% (9/12)
 MYC/BCL2 Coexpress
55% (11/20)
67% (8/12)
 Ki67
20–99% (≥70% in 28/32)
20–99% (≥70% in 60/69)
 Complex karyotype
100% (5/5)
100% (27/27)
Li et al (2015). Modern Pathology 28:

22. Double hit lymphoma: the MD Anderson Cancer Center clinical experience
Survival by baseline characteristics. (A) Event‐free survival and overall survival in the entire cohort. (B) Event‐free survival by histopathology. (C) Event‐free survival by stage. (D) Event‐free survival by age. (E) Event‐free survival by the type of BCL2/BCL6 abnormality. (F) Overall survival after first progression or recurrence. EFS, event‐free survival; OS, overall survival; PFS, progression‐free survival; DLBCL, diffuse large B‐cell lymphoma; FL3, follicular lymphoma grade 3; BCLU, B‐cell lymphoma, unclassifiable with features intermediate between DLBCL and Burkitt lymphoma.
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
British Journal of Haematology Volume 166, Issue 6, pages , 18 JUN 2014 DOI: /bjh

23. Double hit lymphoma: the MD Anderson Cancer Center clinical experience
Survival by treatment. (A) Event‐free survival by initial treatment. (B) Overall survival by initial treatment. (C) Event‐free survival in patients who achieved CR, based on whether frontline stem cell transplant was performed. (D) Overall survival in patients who achieved CR, based on whether frontline stem cell transplant was performed. EFS, event‐free survival; OS, overall survival; RCHOP, rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone; REPOCH, rituximab, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin; RHCVAD/MA, rituximab, hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, alternating with cytarabine plus methotrexate; SCT, stem cell transplantation.
© IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSION' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER.
British Journal of Haematology Volume 166, Issue 6, pages , 18 JUN 2014 DOI: /bjh

24. Summary
In DHL/aDHL only the PS≥2 and bone marrow involvement were independently associated with shorter EFS and OS
Age, stage and # of extranodal sites have minimal impact on the outcome in DHL/aDHL
Specific version of IPI – DHIPI, which includes only PS≥2 and bone marrow involvement (grading from 0-2)
The outcome of DHL patients with current chemotherapeutic approaches is poor, and dismal for refractory or relapsed disease

25. Summary
R-EPOCH, and frontline SCT should be further evaluated in larger studies with longer follow-up
Therapy recommendations - R-EPOCH, with strong consideration for CNS targeting therapy especially when DHIPI ≥1, and strong consideration for frontline SCT in responding patients
Further prospective research is needed to identify additional biological markers and to develop novel therapies
The FISH for MYC, BCL-2 and BCL6 (?) probably should be performed on all CD10+ (GCB) DLBCL lymphomas, in particular with high MYC, BCL2 and Ki67

26. BCLU

27. Clinical Significance of MYC Expression and/or "High-grade" Morphology in Non-Burkitt, Diffuse Aggressive B-cell Lymphomas: A SWOG S9704 Correlative Study
Cook et al. (2014) American Journal of Surgical Pathology. 38(4): , April 2014. 2

28. TABLE 2
Clinical Significance of MYC Expression and/or "High-grade" Morphology in Non-Burkitt, Diffuse Aggressive B-cell Lymphomas: A SWOG S9704 Correlative Study.
Cook, James; MD, PhD; Goldman, Bryan; Tubbs, Raymond; Rimsza, Lisa; Leblanc, Michael; Stiff, Patrick; Fisher, Richard
American Journal of Surgical Pathology. 38(4): , April 2014.
DOI: /PAS
TABLE 2 Clinical Features at Presentation Based on Presence or Absence of MYC Staining by IHC (P-values for All Comparisons >0.05)
© 2014 by Lippincott Williams & Wilkins. Published by Lippincott Williams & Wilkins, Inc. 2

29. TABLE 3
Clinical Significance of MYC Expression and/or "High-grade" Morphology in Non-Burkitt, Diffuse Aggressive B-cell Lymphomas: A SWOG S9704 Correlative Study.
Cook, James; MD, PhD; Goldman, Bryan; Tubbs, Raymond; Rimsza, Lisa; Leblanc, Michael; Stiff, Patrick; Fisher, Richard
American Journal of Surgical Pathology. 38(4): , April 2014.
DOI: /PAS
TABLE 3 Pathologic Features by Morphology
© 2014 by Lippincott Williams & Wilkins. Published by Lippincott Williams & Wilkins, Inc. 2

30. TABLE 4
Clinical Significance of MYC Expression and/or "High-grade" Morphology in Non-Burkitt, Diffuse Aggressive B-cell Lymphomas: A SWOG S9704 Correlative Study.
Cook, James; MD, PhD; Goldman, Bryan; Tubbs, Raymond; Rimsza, Lisa; Leblanc, Michael; Stiff, Patrick; Fisher, Richard
American Journal of Surgical Pathology. 38(4): , April 2014.
DOI: /PAS
TABLE 4 Pathologic Features of Cases With or Without MYC Staining by IHC
© 2014 by Lippincott Williams & Wilkins. Published by Lippincott Williams & Wilkins, Inc. 2

31. Clinical Significance of MYC Expression and/or "High-grade" Morphology in Non-Burkitt, Diffuse Aggressive B-cell Lymphomas: A SWOG S9704 Correlative Study
This study has confirmed the prognostic significance of MYC positivity by IHC in DLBCL and, for the first time, extends this observation to cases of BCLU
BCLU was associated with increased incidence of MYC expression but no distinct clinicopathological features compare to DLBCL
MYC+ cases were morphologically and phenotypically heterogeneous and had poor prognosis (PFS and OS)
MYC IHC is suggested for use in routine clinical practice to assess prognosis in DLBCL
Additional studies utilizing MYC IHC to risk-stratify therapies should also be considered.

32. DHS-0: BCL2 <70% and MYC <40%
Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with R-CHOP
DHS-0: BCL2 <70% and MYC <40%
DHS-1: ether BCL2 or MYC ≥70% and ≥40
DHS-2: both BCL2 and MYC ≥70% and ≥40
Tina Marie Green et al. JCO 2012;30:

33. MYC
BCL2
DHS 1
Tissues stained for MYC protein and BCL2 protein, photographed at ×200 magnification. The MYC staining pattern is distinctly nuclear, whereas staining for BCL2 shows a cytoplasmic pattern. (A and B) Diffuse large B-cell lymphoma (DLBCL) scored as having (A) ≥ 40% MYC-positive lymphoma cells and (B) < 70% BCL2-positive lymphoma cells (B), resulting in a double-hit score (DHS) of 1. No breaks of MYC or BCL2 were detected by fluorescent in situ hybridization (FISH). (C and D) DLBCL scored as having (C) ≥ 40% MYC-positive lymphoma cells and (D) ≥ 70% BCL2-positive lymphoma cells, resulting in a DHS of 2. FISH analysis found breaks of both MYC and BCL2 to be present, making this a case of classic double-hit lymphoma.
DHS 2
Tina Marie Green et al. JCO 2012;30:
©2012 by American Society of Clinical Oncology

34. Overall survival (OS) and progression-free survival (PFS) after treatment with rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone in patients with diffuse large B-cell lymphoma who were divided into groups on the basis of the double-hit s...
Overall survival (OS) and progression-free survival (PFS) after treatment with rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisone in patients with diffuse large B-cell lymphoma who were divided into groups on the basis of the double-hit score (DHS). The 43 patients with a DHS of 0 (DHS 0) and the 88 patients with a DHS of 1 (DHS 1) have similar (A) OS and (B) PFS (P = .416 and P = .876, respectively) and was considered as one group (DHS 0/1) in subsequent analyses. Kaplan-Meier curves of (C) OS and (D) PFS in 54 patients with a DHS of 2 (DHS 2) versus 131 patients in the DHS-0/1 group show that a high DHS is significantly associated with inferior OS (P < .001) and PFS (P < .001). Kaplan-Meier curves of (E) OS and (F) PFS in 35 patients with a DHS-2 score versus 79 patients with DHS-0/1 scores, all from the validation set (VS), confirm that a high DHS is significantly associated with inferior OS (P = .009) and PFS (P < .001).
Tina Marie Green et al. JCO 2012;30:
©2012 by American Society of Clinical Oncology

35. Double Hit Score (DHS) (Tina Marie Green et al. JCO 2012;30:3460-3467))
COO
All patients
(193)
DLBCL
DHL+
DHL-
DHS 0/1
DHS 2
DHS 2 without DHL
GCB type
106 (56%)
10 (91%) *
95 (54%)
84 (65%)
20 (37%)
12 (27%)
ABC type
83 (44%)
1 (9%)
81 (46%)
46 (35%)
34 (63%)
33 (73%)*

36. Double Hit Score (DHS or DPS) (Tina Marie Green et al
Double Hit Score (DHS or DPS) (Tina Marie Green et al. JCO 2012;30: )
DHS system is useful in identifying DLBCL with a “double hit” biology, which is strongly associated with poor prognosis on R-CHOP therapy
DHS 0/1
DHS 2
P value
OS, 3 years
86%
43%
P<0.001
PFS, 3 years
75%
39%
DHS-2 OS and PFS was independent of IPI score and COO
DHS-2 w/o DHL is more common in ABC DLBCL
Include cMYC and Bcl-2 IHC stains on all diffuse large B cell lymphomas
For cMYC we prefer rabbit monoclonal antibody Clone Y69
For Bcl-2 - Clone 124 (Dako) and/or Clone E17 (Epitomics)

37. CD5+ DLBCL
Clinical presentation - elevated serum LDH level, advanced stage, frequent involvement of extranodal sites, presence of B symptoms, and presence of bulky mass, but a sex difference was not found
Gene expression signature is similar in ABC-DLBCL and CD5+ DLBCL
Cyclin D2 was found to be overexpressed in 98% of de novo CD5-positive DLBCL

38. CD5+ DLBCL
Common variant (centroblastic/monomorphic variant) - This variant was the most common -76%
Giant cell rich variant - Giant cells with multiple nuclei intermixed with immunoblasts and centroblasts were observed in 11% of patients. Intravascular involvement is frequent in this variant.
Polymorphic variant – 12% of patients
Immunoblastic variant: Only 1% of patients

39. Cytomorphologic features of four variants of de novo CD5+ DLBCL
Cytomorphologic features of four variants of de novo CD5+ DLBCL. The cells, varying from medium to large in size, are uniform, with a pale basophilic or amphophilic cytoplasm.
Cytomorphologic features of four variants of de novo CD5+ DLBCL. The cells, varying from medium to large in size, are uniform, with a pale basophilic or amphophilic cytoplasm. (A) Common variant, which can be described as the monomorphic or centroblastic variant. Snowman-like, bi-nucleated cells were seen (arrow). (B) Giant cell-rich variant. (C) Polymorphic variant, characterized by polymorphous proliferation with medium and large-sized cells. The immunoblastic variant (D) was rare in our case series.
Motoko Yamaguchi et al. Haematologica 2008;93:
©2008 by Ferrata Storti Foundation

40. Survival according to the histological features of de novo CD5+ diffuse large B-cell lymphoma (DLBCL).
Survival according to the histological features of de novo CD5+ diffuse large B-cell lymphoma (DLBCL). (A) Overall survival in all 120 patients with de novo CD5+ DLBCL. (B) Overall survival of patients with different histological variants of de novo CD5+ DLBCL (C) Patients with the common variant had a better survival than those with the other three variants of de novo CD5+ DLBCL. (D) The presence of intravascular/sinusoidal infiltration had an impact on the overall survival. IVL, intravascular/sinusoidal.
Motoko Yamaguchi et al. Haematologica 2008;93:
©2008 by Ferrata Storti Foundation

41. CD5+ DLBCL – differential Diagnosis
CLL (Richter's Transformation to DLBCL):
about 5–10% of CLL patients
may lose or retain the CD5 molecule
have a different molecular profile than de novo CD5+ BCL6 gene rearrangement is not seen in Richter's
Mantle Cell lymphoma (MCL):
Most cases of MCL over express cyclin D1 and have
t (11; 14)
Secondary CD5+ DLBCL (Non-Richter's): Rarely, CD5 can be acquired in patients with CD5- DLBCL, progression of CD5- low grade B cell lymphoma and CD5+ follicular lymphoma

42. CD5+ DLBCL – differential Diagnosis
Intravascular B cell Lymphoma:
CD5 expression is seen in about 40% cases
Negative for CD29 and CD54
Nonspecific clinical presentation
Primary CNS lymphoma:
30% cases can express CD5
differentiation from de novo CD5+ DLBCL with CNS involvement can be difficult
Analyses on identifying expression of synaptophysin and other CNS specific proteins are ongoing in CD5+ DLBCL

43. Progression-free (A) and overall (B) survival curves of patients with diffuse large B-cell lymphoma according to the presence or absence of CD5.
Progression-free (A) and overall (B) survival curves of patients with diffuse large B-cell lymphoma according to the presence or absence of CD5.
N. Niitsu et al. Ann Oncol 2010;21:
© The Author Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please

44. Overall survival of patients in the chemotherapy group (n = 153) and in the R-chemotherapy group (n = 184).
Overall survival of patients in the chemotherapy group (n = 153) and in the R-chemotherapy group (n = 184).
K. Miyazaki et al. Ann Oncol 2011;22:
© The Author Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please

45. Progression-free (A) and overall (B) survival curves of patients with CD5-positive diffuse large B-cell lymphoma according to whether they received rituximab combination chemotherapy or chemotherapy alone.
Progression-free (A) and overall (B) survival curves of patients with CD5-positive diffuse large B-cell lymphoma according to whether they received rituximab combination chemotherapy or chemotherapy alone. NS, not significant.
N. Niitsu et al. Ann Oncol 2010;21:
© The Author Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please

46. (A) Overall survival (OS) curves of patients with germinal center B-cell type CD5-positive (CD5+) diffuse large B-cell lymphoma or nongerminal center B-cell type CD5+ diffuse large B-cell lymphoma.
(A) Overall survival (OS) curves of patients with germinal center B-cell type CD5-positive (CD5+) diffuse large B-cell lymphoma or nongerminal center B-cell type CD5+ diffuse large B-cell lymphoma. (B) OS curves according to the therapeutic method among the patients with CD5+ diffuse large B-cell lymphoma. CHOP, combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone; CyclOBEAP, cyclophosphamide, vincristine, bleomycin, etoposide, doxorubicin, and prednisone; GCB, germinal center B-cell-like group.
N. Niitsu et al. Ann Oncol 2010;21:
© The Author Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please

47. CD5+ DLBCL - Summary
De novo CD5+ DLBCL is clinico-pathologically and genetically distinct from CD5-negative DLBCL and MCL lymphoma.
Accounts for 5-10% of all DLBCL
Has an aggressive course and high IPI score at presentation
GEP is similar to ABC type of DLBCL (83% are of ABC type)
Rituximab-based therapy improves PFS but does not decrease CNS relapse rate or OS
CD5 stain should be performed on all cases of DLBCL

48. Summary on intrinsic biology of DLBCL
Determine:
COO
CD5 on all DLBCL
MYC, BCL2 and Ki67 on all lymphoma regardless of morphology
FISH for MYC, BCL2 and BCL6 on DLBCL with high expression of MYC, BCL2, Ki67 and CD10

49. Prognostic Markers in DLBCL
Absolut Monocyte Count (AMC) and Absolute Lymphocyte Count (ALC) Prognostic Index:
Low - AMC <610/µl and ALC >1000/µl
Intermediate – AMC ≥ 610/µl or ALC > 1000/µl
High - AMC ≥610/µl and ALC ≤1000/µl

50. Absolut Monocyte Count (AMC) and Absolute Lymphocyte Count (ALC) Prognostic Index
By univariate analysis, the AMC/ALC score was a predictor for OS and PFS.
On multivariate analysis performed including the cell of origin (COO) and the International Prognostic Index, AMC/ALC score remained an independent predictor for OS and PFS.
The AMC/ALC score was able to further stratify DLBCL clinical outcomes by COO
The AMC/ALC score was independent of COO and added to its ability to identify patients with high-risk disease
This prognostic score was independent of the IPI and added to its ability to identify high-risk patients

51. Kaplan–Meier estimates of progression-free (a, b) and overall (c, d) survival for the entire cohort of patients stratified by the AMC/ALC prognostic score (a, c) and the International Prognostic Index (b, d) are shown.
R A Wilcox et al. Leukemia (2011) 25, 1502–1509

52. Macrophage Polarization immunophenotype
Classic activation – M1 phenotype
CD68, CD80, CD86, HLA-DR, TNFα, IL-12, CCR7…
Alternative activation – M2 and M2-like
CD68, CD163, CD206, IL10, CCL22 …
*CD68 – KP1 and PG-M1 clones

54. Staining of M2 TAM by CD163

55. Tumour‐associated macrophages in diffuse large B‐cell lymphoma: a study of the Osaka Lymphoma Study Group (2-year survival rate)
91.9%
82.1%
61%
60%
 The estimated 2‐year survival rate in the high total tumour‐associated macrophage (TAM) group was 60.0%, and that in the low total TAM group was 82.1%. The overall survival rate in the high total TAM group was significantly worse than that in the low total TAM group (P < 0.05).
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Histopathology Volume 60, Issue 2, pages , 23 DEC 2011 DOI: /j x

56. An increase of M2 macrophages predicts poor prognosis in patients with diffuse large B-cell lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone Nam et al. Leuk Lymphoma :

57. Prognostic implication of types of tumor-associated macrophages in Hodgkin lymphoma
Virchows Archiv 2011; 459:

58. Tumour‐associated macrophages in lymphomas
Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment.
An increase in CD68 (+) cells was related to improved overall survival (OS). By contrast, an increased number of CD163 (+) cells and a higher ratio of CD163+/CD68+ cells were significantly associated with shorter OS and progression-free survival (PFS).
In multivariate analysis, an increased ratio of CD163+/CD68+ cells was an independent predictor of shorter OS and PFS.
These results suggest that M2 macrophages might have a lymphoma-promoting function in DLBCL and predict poor clinical outcome.
Therapeutic approaches targeting M2 macrophages would be valuable for the management of DLBCL in the R era.

59. 76 Patients North Central Cancer Treatment Group trial N0489
Elevated Serum Free Light Chains Are Associated With Event-Free and Overall Survival in Two Independent Cohorts of Patients With Diffuse Large B-Cell Lymphoma
76 Patients North Central Cancer Treatment Group trial N0489
219 Patients UIHC/Mayo Clinic Specialize Program of Research Excellence Molecular Epidemiology Resource (MER)
- Abnormal ƙ/ƛ or elevated FLC in 32% and 14%
- Elevate FLC was the strongest predictor of outcome in multivariable models with the IPI components
Increased serum FLC is an independent adverse prognostic factor for EFS and OS in DLBCL

60. (A) Event-free survival and (B) overall survival Kaplan-Meier survival curves by serum free light chain (FLC) in two cohorts (North Central Cancer Treatment Group trial N0489 and the Specialized Program of Research Excellence Molecular Epidemiology Resource...
(A) Event-free survival and (B) overall survival Kaplan-Meier survival curves by serum free light chain (FLC) in two cohorts (North Central Cancer Treatment Group trial N0489 and the Specialized Program of Research Excellence Molecular Epidemiology Resource [MER]) of patients with untreated diffuse large B-cell lymphoma.
Maurer M J et al. JCO 2011;29:
©2011 by American Society of Clinical Oncology

61. Free Light Chains
Kappa and Lambda measurements were similar: Elevated FLC Abnormal κ:λ
NO489 (n=79) 34% 12%
MER (n=219) 31% 15%
Patients with elevated FLC – inferior OS and EFS (P<.001)
The result remain significant for EFS and OS after adjusting for IPI (in all IPI scores – independent of IPI!)
Abnormal κ:λ ratio was modestly associated with outcome (P<.07) probably due to elevated FLC alone
Patients with normal range of κ and λ, the abnormal κ:λ ratio was not associated with outcomes (P=0.99)

62. Outcome by type of free light chain (FLC) abnormality.
Outcome by type of free light chain (FLC) abnormality. (A) Event-free survival; (B) overall survival.
Maurer M J et al. JCO 2011;29:
©2011 by American Society of Clinical Oncology

63. Elevated Monoclonal Free Light Chains Are a Serum Marker of ABC Type Diffuse Large B-Cell Lymphoma
Lambda Quantitative Free Light Chains mg/L
Kappa Quantitative Free Light Chains mg/L
Kappa/Lambda Free Light Chain Ratio

64. ~10% of DLBCL patients have elevated monoclonal sFLC with poor outcome
Elevated Monoclonal Free Light Chains Are a Serum Marker of ABC Type Diffuse Large B-Cell Lymphoma
~10% of DLBCL patients have elevated monoclonal sFLC with poor outcome
The great majority of patients (10/11 or 91%) with elevated monoclonal sFLC have an ABC-type DLBCL by Tally algorithm
Secretion of FLC by tumor suggests an activated B cell clone (characteristic for ABC type DLBCL)
Following the sFLC may monitor response to therapy and progression

65. Prognostic Markers in DLBCL
BCL6 expression
BCL6+ better responded to CHOP than BCL6-
Therapy with R-CHOP benefitted BCL6- DLBCL but did not changed the response for BCL6+ DLBCL
P53+++/P21- as a surrogate for p53 mutation,
inferior survival in GBC but not in ABC DLBCL
P21+ - favorable independent marker in R-CHOP but not CHOP treated patients
BCL2+ - benefit for R-CHOP regimen for PFS and OS
- BCL2- show benefit in PFS but not in OS on R-CHOP

66. MCL

67. MIPI score = [0.03535 × age (years)] × age (years)]
(if ECOG > 1)
+ [1.367 × log10(LDH/ULN)]
+ [ × log10(WBC count)]
LR - <5.7; IR – 5.7-<6.2; HR - >6.2
Simplified MIPI
Points
Aga, y
ECOG
LDH/ULN
WBC, 109/L
<50
0-1
<0.67
<6.700 1
50-59 - 2
60-69
2-4 3
≥70
≥1.5
≥15.000
Biological MIPI = MIPI score x Ki-67 (%)
LR - <5.7; IR – 5.7-<6.5; HR - >6.5

68. MIPI score = [0. 03535 × age (years)] × age (years)] + 0
MIPI score = [ × age (years)] × age (years)] (if ECOG > 1) + [1.367 × log10(LDH/ULN)] + [ × log10(WBC count)]

69. Histopathology, cell proliferation indices and clinical outcome in 304 patients with MCL (European MCL Network) Tiemann M at al British J Haematology 131:29-38

70. Kaplan-Meier plot for overall survival of patients treated with CHOP (A) and R-CHOP (B) stratified in 3 groups according to the Ki-67 index of less than 10% (< 10), 10% to less than 30% (≥10), and 30% or more (≥30) Ki-67 positive cells.
Kaplan-Meier plot for overall survival of patients treated with CHOP (A) and R-CHOP (B) stratified in 3 groups according to the Ki-67 index of less than 10% (< 10), 10% to less than 30% (≥10), and 30% or more (≥30) Ki-67 positive cells.
Olaf Determann et al. Blood 2008;111:
©2008 by American Society of Hematology

71. * * * Hot spots > Proliferating T cells Residual GC
Klapper et al (2009). Ki-67 as a prognostic marker in mantle cell lymphoma—consensus guidelines
of the pathology panel of the European MCL Network. J Hematopathology 2:
Residual GC
* Hot spots
> Proliferating T cells * *

72. SOX11 and TP53 add prognostic information to MIPI in a homogenously treated cohort of mantle cell lymphoma – a Nordic Lymphoma Group study
Representative immunostainings of CCND1 and TP53 and correlation of TP53 to overall survival and event‐free survival. (A) Weak CCND1 staining was detected in 6% of cases, (B) intermediate CCND1 staining was detected in 34% of cases, and (C) strong CCND1 staining was detected in 60% of cases. (D) Weak TP53 staining was detected in 84% of cases, (E) intermediate TP53 staining was detected in 5% of cases and (F) strong TP53 was detected in 11% of cases. Survival analysis using Kaplan‐Meier's method comparing p53weak (F), p53intermediate (E) and p53strong (F) shows a negative correlation between TP53 and (G) overall survival and (H) event‐free survival with P‐values <0·001 in both cases, as determined by the log rank test.
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British Journal of Haematology Volume 166, Issue 1, pages , 29 MAR 2014 DOI: /bjh

73. MCL – Summary
IHC – CD3, CD5, CD20, CD23, Cyclin D1, SOX11, MIB1 (Ki-67), TP53
SOX11 – “low” and “high”
TP53 – negative, weak, intermediate (strong stain <30%), strong (strong stain in >30%)
Ki-67 index:
On primary diagnosis before treatment
Representative area which do not include GC, hot spots of proliferation and proliferating T cells
2 x 100 cells show high concordance with a gold standard (2 x 500)
For reporting use a cutoff of <30% or >30%

74. Follicular Lymphoma and FLIPI scoring system
FLIPI - Age >60 years, Stage III or IV disease, >4 lymph node groups involved, Serum Hb <12 g/dL and Elevated serum LDH
The OS rate is 75% at 5 years, and ranges from 71% at 10 years for patients with a FLIPI score between 0 to 1, and 36% for those with a FLIPI score of >3.
Median survival of all newly diagnosed patients is approximately 9 years. Although the OS appears to have improved with advances in chemoimmunotherapy, the median event free survival (EFS) remains around 2 years for patients with advanced disease.
FLIPI has several limitations. It focuses on clinical factors and does not take into account biological factors such as the tumor microenvironment and the host response
When some patients with FL surviving <1 year and others >20 years, additional prognostic indicators are clearly needed to refine risk adapted therapy.
FLIPI score
Risk category
% of patients
Median PFS (m)
0 or 1
Good 36 84 2
Inter­mediate 37 70
3 - 5
Poor 27 42

75. Elevated serum levels of IL-2R, IL-1RA, and CXCL9 are associated with a poor prognosis in follicular lymphoma
Serum cytokines and chemokines may reflect tumor biology and host response in follicular lymphoma (FL).
30 cytokines and chemokines were measured in pretreatment serum specimens from newly diagnosed FL patients (n = 209) and from 400 matched controls.
Cytokine levels were correlated with clinical outcome in patients who were observed or received single agent rituximab, or those who received chemotherapy.

76. Elevated serum levels of IL-2R, IL-1RA, and CXCL9 are associated with a poor prognosis in follicular lymphoma
Six cytokines were associated with outcome in the Molecular Epidemiology Resource (MER) after adjusting for the FL international prognostic index.
In patients who were observed or treated with rituximab alone, increased serum IL-12 and interleukin 1 receptor antagonist (IL-1RA) (P = .005 and .02) were associated with a shorter event-free survival.
In patients receiving chemotherapy, hepatocyte growth factor, IL-8, IL-1RA, and CXCL9 (P = .015, .048, .004, and .0005) predicted a shorter EFS.
When the MER chemotherapy treated patients and SWOG (n=183) patients were combined in a meta-analysis, IL-2R, IL-1RA, and CXCL9 (P = .013, .042, and .0012) were associated with a poor EFS.

77. EFS by cytokine in all MER patients
EFS by cytokine in all MER patients. The EFS by serum IL-12 (A), serum IL-2R (B), IL-1RA (C), and CXCL9 (D) levels for all patients in the MER cohort.
Muhammad A. Mir et al. Blood 2015;125:
©2015 by American Society of Hematology

78. EFS by cytokine in MER subgroups
(A – observed or received rituximab; B-D – treated with chemotherapy)
EFS by cytokine in MER subgroups. The EFS by serum IL-12 levels in patients in the MER cohort who were observed or received rituximab monotherapy (A). The EFS by serum IL-2R (B), CXCL9 (C), and IL-1RA (D) levels in patients in the MER cohort who were treated with chemotherapy.
Muhammad A. Mir et al. Blood 2015;125:
©2015 by American Society of Hematology

79. Conclusions The IPI does not capturer the biologic spectrum of DLBCL
COO (by molecular or IHC) subtyping doesn’t identify patients with highly aggressive DLBCL since these patients will be in both subgroups
For COO by IHC choose most convenient algorithm
The FISH for MYC, BCL-2 and BCL6 should be performed on all CD10+ (GCB) DLBCL lymphomas
BCLU (features of DLBCL and BL) with high MYC (>40%) and Ki67 (>75%) should lead to FISH to exclude a DHL or aDHL.
Report primary CD5+ DLBCL
MCL - for Ki67 reporting use a cutoff of <30% or >30%

80. DLBCL, centroblastic variant

81. DLBCL, anaplastic variant

82. DLBCL with clear cytoplasm

83. DLBCL, T-cell/histiocyte-rich type

84. DLBCL, Hodgkin-like (Lymph node)

85. DLBCL, multilobated variant

86. DLBCL, multilobated variant

87. Intravascular large B cell lymphoma
Involving the lumens of small vessels
Widely disseminated at presentation and may
present with skin lesions, neurologic symptoms
Lack expression of CD29 and CD54
Extremely aggressive clinical course
Kidney
Lung
CD20

88. Plasmablastic lymphoma

89. Primary mediastinal large B-cell lymphoma

90. DLBCL, immunoblastic variant

91. BCLU

92. MCL, common variant

93. MCL, interfollicular pattern

94. MCL, small cell/diffuse variant (SLL-like)
Pink
Histiocytes

95. MCL, pleomorphic variant

96. MCL, blastoid variant

97. ?
MCL CD23+
MCL CD23+

98. ?
MCL CD23+
MCL CD23+

99. ?
MCL CD23+
MCL CD23+

100. ?
MCL CD23+
MCL CD23+

101. CD5
CD3
CD23
CD20
MCL CD23+

102. Cyclin D+ MCL CD5+/CD23+
MCL CD23+

103. MCL, nodal variant
MCL, nodal variant

104. 20X
CD3
CD20
Bcl-6

105. 60x
CD10
Bcl-2
CD5

106. Cyclin D1
CD5- MCL, nodal variant