1. Diagnostically and Prognostically Significant Genetic Alterations in Diffuse Large B-Cell Lymphoma Friederike Kreisel, MD Department of Pathology and Immunology Washington University in St. Louis

2. Frequency of B-and T-Cell Lymphomas 33% 22.1%9.4% 6.7% 11.7% 6.0% 2.5% 8.6% Incidence of non-Hodgkin lymphoma (NHL) has increased more than 73% between 1973 and 1991 Estimated rate for DLBCL is ~4.68 cases per 100,000 persons/year

3. Normal B-Cell Differentiation NEJM 2000, 343;108-117

4. The Germinal Center Germinal Center NEJM 2000, 343;108-117 Lymph node CD10+/BCL-6+/BCL-2-

5. Diffuse Large B-Cell Lymphoma Usually de novo (primary) Transformation from: –CLL/SLL –Follicular lymphoma Immunodeficiency is strongest known risk factor WHO 2008, Tumors of Hematopoietic and Lymphoid Tissues

6. Diffuse Large B-Cell Lymphoma Clinically, morphologically, and genetically heterogenous group with only a subset falling into the WHO subcategories 20-30% of DLBCL continue to be defined by their nuclear size only

7. DLBCL Subcategories (WHO 2008) Diffuse large B-cell lymphoma, NOS T-cell/histiocyte rich DLBCL Primary DLBCL of CNS Primary cutaneous DLBCL, leg type EBV positive DLBCL of the elderly Primary mediastinal (thymic) LBCL Intravascular LBCL DLBCL associated with chronic inflammation Lymphomatoid granulomatosis ALK-positive LBCL Plasmablastic lymphoma LBCL arising in HHV8+ multicentric Castleman’s disease Primary effusion lymphoma B-cell lymphoma, unclassifiable

8. International Prognostic Index (IPI) most commonly used to predict outcome in DLBCL Unfavorable variables –Age >60 years –Poor performance status (ECOG  2) –Advanced Ann Arbor stage (III-IV) –Extranodal involvement  2 sites –High serum LDH (>normal) Risk groupUnfavorable variables All patients Patients  60 Low 0 or 1 0 Low/intermediate 2 1 High/intermediate 3 2 High 4 or 5 3

9. Major Recurring Genetic Events in DLBCL Genetic DefectFrequencyLocation BCL635-40%3q27 BCL2t(14;18) 13%, amplification 24% 18q21 cMYC15%8q24 FAS(CD95)20%10q24 Aberrant SHM45% Genes aberrantly targeted: BCL6, cMYC, PAX5 TP5316%17p

10. BCL6 Zinc-finger transcription repressor normally expressed within germinal center (GC) B-cells  BCL6 null animals fail to generate GCs in response to antigen Constitutive expression of BCL6 might  the p53- mediated apoptosis, promoting persistence of malignant clones Wikipedia

11. BCL2 Proto-oncogene located at 18q21 that promotes B-cell survival via inhibition of apoptosis and confers chemotherapy resistance BCL-2 expression is normally down-regulated in the GC where apoptosis plays a critical role in negative B-cell selection t(14;18)  fusion gene leading to transcription of  levels of BCL2 Wikipedia

12. Gene Expression Profiling Measurement of the activity (expression) of thousands of genes at once  relative amount of mRNA expressed  provides a global picture of cellular function

13. Gene Expression Profiling Highlight similarities between subsets of DLBCL and normal B cells Define robust and highly reproducible DLBCL subtypes with comprehensive transcriptional signatures Identify features associated with unfavorable responses to empiric combination chemotherapy

14. DNA Microarray - Methodology + + + + + – – – – DLBCL2 DLBCL1 LYMPHOCHIP Slides are coated with poly- Lysine, which is positively charged. DNA is negatively charged, so it “sticks” to the slide through ionic inter- action cDNA is made from different DLBCL tumors LYMPHOCHIP

15. DNA Microarray - Methodology DLBCL2 DLBCL1 LYMPHOCHIP Both sets of DLBCL cDNA are labelled with different fluorescent tags, in this case red and green LYMPHOCHIP DLBCL1 DLBCL2 Gene array lymphochip is incubated with the tagged DLBCL cDNAs, which bind to the matching genes printed on the array

16. DNA Microarray - Methodology LYMPHOCHIP DLBCL1 DLBCL2 Since positions of the genes on the DNA array are known, levels of gene expression can be figured out based on color signal. If the gene is only expressed in DLBCL1, the square is red; if the gene is only expressed in DLBCL2, the square is green; if the gene is equally expressed in both DLBCL, the square is yellow.

17. Gene Expression Profiling in DLBCL (Alizadeh et al., Nature 2000) Ratio of hybridization of fluorescent experimental mRNA to fluorescent pooled reference mRNA

18. Gene Expression Profiling in DLBCL (Alizadeh et al., Nature 2000) a.Hierarchical clustering of DLBCL (orange and blue) and germinal center B cells (black) based on the genes of the germinal center B-cell gene expression signature b.Discovery of genes that are selectively expressed in GC B-like DLBCL and activated B-like DLBCL based on genes of pan B-cells, germinal center B-cells, and activated B-cells c.Hierarchical clustering of the genes selectively expressed in GC B-like DLBCL and activated B-like DLBCL, which was determined from Fig 3b

19. Differences Between GC-like and Activated B-like DLBCL GC B-like DLBCLActivated B-like DLBCL Cell of originGerminal center B-cellPostgerminal center B- cell Chromosomal alterations t(14;18); gains of 12qGains of 3q and 18q21-22; deletion of 6q21-q22, BCL6 translocations Oncogenic mechanisms BCL2 translocation; amplification of cREL locus Constitutive NFkB activation Ongoing Ig mutation YesNo IHCExpression of CD10, BCL-6Expression of MUM1 Clinical outcomeFavorable (60% 5-year survival) Poor (35% 5-year survival)

20. Overall survival of DLBCL patients grouped on the basis of gene profiling shows worse outcome for patients with “activated B-like” subtype A.A. Alizadeh et al. Nature 2000; 403:503-511

21. Immunohistochemistry (IHC) can be used to determine the GC B-like and Activated B-like subtypes of DLBCL Normal Lymph Node GC B-like DLBCL Activated B-like/non-GC B-like DLBCL C.P. Hans et al. Blood 2004, 103:275-282 CD10BCL-6

22. Overall survival curves comparing immunohistochemical and genetic classification of GC B-like and activated B-like DLBCL are worse for the activated B-like subtype C.P. Hans et al. Blood 2004, 103:275-282

23. Decision Tree for IHC Classification of DLBCL Sensitivity of IHC 71% for the GC B-like group and 88% for the activated B-like / non GC B-like group PPV of IHC was 87% for the GC B-like group and 73% for the activated B-like / non GC B-like group 30/142 (21%) of cases had discordant IHC and cDNA microarray results C.P. Hans et al. Blood 2004, 103:275-282

24. Does the type of therapy for DLBCL affect prognosis in patients? Drugs in CHOP: –C = Cyclophosphamide –H = Doxorubicin (Hydroxydaunomycin) –O = Vincristine Sulfate (Oncovin) –P = Predisone Drugs in R-CHOP (since 2000): –R = Rituximab –C = Cyclophosphamide –H = Doxorubicin (Hydroxydaunomycin) –O = Vincristine Sulfate (Oncovin) –P = Predisone

25. Rituximab CD20 antigen –Expressed only on B-cells –Present in >90% of B-cell lymphomas –Does not shed off cell surface –Important for cell cycle initiation and differentiation B-cell Murine antigen binding domain Human IgG1 constant region Human k constant region CD20

26. Effects of Rituximab Mediates antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity Elicits shedding of CD23 Down-regulates the B-cell receptor Induces apoptosis of CD20+ cells Half-life of 30-400 hours (varies by dose and length of treatment)

27. OS Curves of Patients with GC B-Like and Activated B-like DLBCL by IHC After CHOP or R-CHOP Treatment R. Seki et al. Cancer Sci 2009, 100:1842-47

28. The overall survival rates for control and immunochemotherapy-treated DLBCL patients according to immunohistochemical and clinical factors H. Nyman et al. Blood 2007;109:4930-4935 CHOPR-CHOP

29. The Hans Classifier – Controversies in the CHOP and R-CHOP Era Hans et al. Blood 2004 Berglund et al. Mod Path 2005 Haarer et al. Arch Pathol 2006 Muris et al. J Pathol 2006 Sjo et al. Eur J Hematol 2007 Van Imhoff et al. J Clin Oncol 2007 Nyman et al. Blood 2007 Amara et al. Mod Pathol 2008 Fu et al. JCO 2008 (R-CHOP) Colomo et al. Blood 2003 Nyman et al. Blood 2007 (R-CHOP) De Paepe et al. JCO 2005 Dupuis et al. Haematologica 2007 Natkunam et al. JCO 2008 Veelken et al. Ann Oncol 2007 Amen et al. Histopathology 2007 Wilson et al. JCO 2008 (DE-EPOCH-R) Ott et al. Leukemia Research 2012 (R-CHOP) Survival association present Survival association absent

30. Transcriptional Profile of DLBCL Subsequently Treated with R-CHOP J.P. Jais et al. Leukemia 2008;22:1917-24

31. The recognition of GCB and ABC DLBCL is highly reproducible by gene expression profiling between different centers GCBUnclassABC GCB5330 Unclass3162 ABC0348 Center A Center B Concordant results in 117/128 (91%) Unpublished data from Elias Campo, MD

32. Prognostic Impact of Gene Alterations in DLBCL in the Era of Rituximab MYC gene rearrangements CDKN2A (p16INK4/p14ARF) and CDKN2B (p15INK4) deletions p53 aberrations

33. cMYC Located on 8q24 Regulates gene expression through binding on Enhancer Box sequences and recruiting histone acetyl-transferases Burkitt lymphoma t(8;14) - cMYC gene next to the immunoglobulin heavy chain locus  fusion gene causing overexpression of MYC proto- oncogene in lymphoma cells Wikipedia

34. Outcomes of Patients with MYC + or MYC - DLBCL 135 cases of DLBCL analyzed 12/134 cases (8.8%) were positive for MYC rearrangement All patients were treated with R-CHOP K.J. Savage et al. Blood 2009; 114:3533-37

35. CDKN2A/B (p16,p14,p15) Located on 9p21.3 Tumor suppressor gene that regulates cell cycle via the retinoblastoma and p53 apoptosis pathways Defective p53/CDKN2A/B signaling can lead to apoptosis resistance in DLBCL Kreisel et al. Cancer Genetics 2010;204:129-37

36. TP53 Located on 17p13.1 Tumor suppressor gene “The guardian of the genome” p53 is in reference to its molecular weight (53-kDa protein) 50% of human tumors contain a mutation or deletion of the TP53 gene Wikipedia

37. OS according to the TP53 and CDKN2A allelic status in DLBCL. (A) Overall survival (OS) probability in the entire lymphoma population (n = 114). (B) OS probability in the R-CHOP treated subgroup (n=78) Jardin F et al. Blood 2010;116:1092-1104

38. Schematic representation of the 9p21 locus and its genomic alterations detected by a dedicated quantitative multiplex PCR of short fragments (QMPSF) assay in DLBCL. Jardin F et al. Blood 2010;116:1092-1104 p16p14p15 Gray: heterozygous deletion Black: homozygous deletion CDKN2A/B

39. Summary DLBCL represents a morphologically, biologically, and clinically heterogenous group of tumors ~20-30% of DLBCL cannot be subcategorized into a current WHO subclassification Despite DLBCL subcategories clinical outcome within a specific category is heterogenous Genetic alterations of BCL-6, BCL-2, and c-MYC are among the most common recurrent genetic abnormalities

40. Summary Gene expression profiling of DLBCL offered –Comparison of gene signatures between subsets of DLBCL and normal B cells –Delineation of robust and highly reproducible DLBCL subtypes with comprehensive transcriptional signatures (GC-like vs. activated-like DLBCL) –Identification of gene expression signatures associated with unfavorable response to empiric combination chemotherapy

41. Summary Immunohistochemical sub-categorization into GC-like vs. non GC B-type in Rituximab era remains controversial, although robust with cDNA microarray Additionally, c-MYC, TP53, and CDKN2A/B aberrations appear to have prognostic significance in Rituximab era

42. Journal Club 4/10/12

44. Aim of study Show that the variability in natural history of DLBCL reflects unrecognized molecular heterogeneity in the tumors Measure the activity (expression) of thousands of genes at once (DNA microarrays) Identify molecularly distinct subgroups of DLBCL Correlate different subgroups with clinical outcome

45. Construction of DNA microarray “Lymphochip”: selection of genes that are preferentially expressed in lymphoid cells and genes with known or suspected roles in immunology or cancer –12,069 out of 17,856 cDNA from a germinal center B- cell library –2,338 cDNA from libraries derived from DLBCL, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia –cDNA from genes that are induced or repressed during B- and T-cell-lymphocyte activation by mitogens or cytokines – 3,186 genes of importance to lymphocyte and/or cancer biology

46. cDNA Microarray - Methodology 17,856-GENE ARRAY LYMPHOCHIP 17,856-GENE ARRAY LYMPHOCHIP Experimental sample Reference pool of 9 different lymphoma cell lines The fluorescence ratio was quantified for each gene and reflected the relative abundance of the gene in each experimental mRNA sample compared to the reference pool Experimental sample Reference pool of 9 different lymphoma cell lines

47. Figure 1 dendrogram Each row: separate cDNA clone on the microarray Each column: separate experimental mRNA sample Results: ratio of hybridization of fluorescent experimental mRNA to fluorescent pooled reference mRNA

48. Figure 2

49. “Lymph node survival signature”  host response to lymphoma, is associated with  survival times “Proliferation signature”, a quantitative measurement of tumor cell proliferation, confers an inferior survival “Germinal center B-cell signature” with BCL-6 and CD10 expression

50. Figure 3

51. Figure 4

52. Figure 5