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

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.

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

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

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)

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 < .0001 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 < .0001 for each cohort. Gilles Salles et al. Blood 2011;117:7070-7078 ©2011 by American Society of Hematology

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!

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

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 2014 123:1214-17

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;123:1214-1217 ©2014 by American Society of Hematology

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 2014 123:1214-17

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;29:200-207

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;29:200-207 ©2011 by American Society of Clinical Oncology

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;29:200-207 ©2011 by American Society of Clinical Oncology

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;29:200-207 ©2011 by American Society of Clinical Oncology

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.

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

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

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

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.

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 28:208-217

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 PERMISSIONS@WILEY.COM 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 891-901, 18 JUN 2014 DOI: 10.1111/bjh.12982 http://onlinelibrary.wiley.com/doi/10.1111/bjh.12982/full#bjh12982-fig-0001

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 PERMISSIONS@WILEY.COM 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 891-901, 18 JUN 2014 DOI: 10.1111/bjh.12982 http://onlinelibrary.wiley.com/doi/10.1111/bjh.12982/full#bjh12982-fig-0003

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

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

BCLU

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):494-501, April 2014. 2

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):494-501, April 2014. DOI: 10.1097/PAS.0000000000000147 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

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):494-501, April 2014. DOI: 10.1097/PAS.0000000000000147 TABLE 3 Pathologic Features by Morphology © 2014 by Lippincott Williams & Wilkins. Published by Lippincott Williams & Wilkins, Inc. 2

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):494-501, April 2014. DOI: 10.1097/PAS.0000000000000147 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

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.

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;30:3460-3467

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;30:3460-3467 ©2012 by American Society of Clinical Oncology

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;30:3460-3467 ©2012 by American Society of Clinical Oncology

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%)*

Double Hit Score (DHS or DPS) (Tina Marie Green et al Double Hit Score (DHS or DPS) (Tina Marie Green et al. JCO 2012;30:3460-3467) 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)

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

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

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;93:1195-1202 ©2008 by Ferrata Storti Foundation

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;93:1195-1202 ©2008 by Ferrata Storti Foundation

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

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

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;21:2069-2074 © The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

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;22:1601-1607 © The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com

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;21:2069-2074 © The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

(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;21:2069-2074 © The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

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

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

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

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

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

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

Staining of M2 TAM by CD163

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). 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 PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' 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. Histopathology Volume 60, Issue 2, pages 313-319, 23 DEC 2011 DOI: 10.1111/j.1365-2559.2011.04096.x http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2559.2011.04096.x/full#f3

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. 2014 55:2466-76

Prognostic implication of types of tumor-associated macrophages in Hodgkin lymphoma Virchows Archiv 2011; 459:361-366

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.

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

(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;29:1620-1626 ©2011 by American Society of Clinical Oncology

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)

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;29:1620-1626 ©2011 by American Society of Clinical Oncology

Elevated Monoclonal Free Light Chains Are a Serum Marker of ABC Type Diffuse Large B-Cell Lymphoma Lambda Quantitative Free Light Chains 5.7 - 26.3 mg/L Kappa Quantitative Free Light Chains 3.3 - 19.4 mg/L Kappa/Lambda Free Light Chain Ratio 0.26 - 1.65

~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

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

MCL

MIPI score = [0.03535 × age (years)] × age (years)] + 0.6978 (if ECOG > 1) + [1.367 × log10(LDH/ULN)] + [0.9393 × 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 - 0.67-0.99 6.700-9.999 2 60-69 2-4 1.0-1.49 10.000-14.999 3 ≥70 ≥1.5 ≥15.000 Biological MIPI = MIPI score + 0.02142 x Ki-67 (%) LR - <5.7; IR – 5.7-<6.5; HR - >6.5

MIPI score = [0. 03535 × age (years)] × age (years)] + 0 MIPI score = [0.03535 × age (years)] × age (years)] + 0.6978 (if ECOG > 1) + [1.367 × log10(LDH/ULN)] + [0.9393 × log10(WBC count)]

Histopathology, cell proliferation indices and clinical outcome in 304 patients with MCL (European MCL Network) Tiemann M at al. 2005 British J Haematology 131:29-38

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;111:2385-2387 ©2008 by American Society of Hematology

* * * 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 2:103-111 Residual GC * Hot spots > Proliferating T cells * *

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. 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 PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' 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 1, pages 98-108, 29 MAR 2014 DOI: 10.1111/bjh.12854 http://onlinelibrary.wiley.com/doi/10.1111/bjh.12854/full#bjh12854-fig-0002

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%

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

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.

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.

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;125:992-998 ©2015 by American Society of Hematology

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;125:992-998 ©2015 by American Society of Hematology

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%

DLBCL, centroblastic variant

DLBCL, anaplastic variant

DLBCL with clear cytoplasm

DLBCL, T-cell/histiocyte-rich type

DLBCL, Hodgkin-like (Lymph node)

DLBCL, multilobated variant

DLBCL, multilobated variant

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

Plasmablastic lymphoma

Primary mediastinal large B-cell lymphoma

DLBCL, immunoblastic variant

BCLU

MCL, common variant

MCL, interfollicular pattern

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

MCL, pleomorphic variant

MCL, blastoid variant

? MCL CD23+ MCL CD23+

? MCL CD23+ MCL CD23+

? MCL CD23+ MCL CD23+

? MCL CD23+ MCL CD23+

CD5 CD3 CD23 CD20 MCL CD23+

Cyclin D+ MCL CD5+/CD23+ MCL CD23+

MCL, nodal variant MCL, nodal variant

20X CD3 CD20 Bcl-6

60x CD10 Bcl-2 CD5

Cyclin D1 CD5- MCL, nodal variant