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

Frequency of B-and T-Cell Lymphomas

Diffuse Large B-cellLymphoma

Follicular Lymphoma

Marginal Zone B-cellLymphoma

CLL/SLL

Mantle CellLymphoma

Burkitt Lymphoma

T-cell Lymphomas

Other Types

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 1991Estimated rate for DLBCL is ~4.68 cases per 100,000 persons/year

Normal B-Cell Differentiation

NEJM 2000, 343;108-117

The Germinal Center

Germinal Center

NEJM 2000, 343;108-117

Lymph node

CD10+/BCL-6+/BCL-2-

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

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

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

International Prognostic Index (IPI) most

commonly used to predict outcome in DLBCL • Unfavorable variables

– Age >60 years– Poor performance status (ECOG2)– Advanced Ann Arbor stage (III-IV)– Extranodal involvement 2 sites– High serum LDH (>normal)

Risk group Unfavorable variables

All patients Patients60

Low 0 or 1 0

Low/intermediate 2 1

High/intermediate 3 2

High 4 or 5 3

Major Recurring Genetic Events in DLBCL

Genetic Defect Frequency Location

BCL6 35-40% 3q27

BCL2 t(14;18) 13%, amplification 24%

18q21

cMYC 15% 8q24

FAS(CD95) 20% 10q24

Aberrant SHM 45% Genes aberrantly targeted: BCL6,

cMYC, PAX5

TP53 16% 17p

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

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

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

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

DNA Microarray - Methodology

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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 DLBCLtumors

LYMPHOCHIP

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 incubatedwith the tagged DLBCL cDNAs, whichbind to the matching genes printed on the array

DNA Microarray - Methodology

LYMPHOCHIP

DLBCL1DLBCL2

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 geneis only expressed in DLBCL2, the square is green; if the gene isequally expressed in both DLBCL, the square is yellow.

Gene Expression Profiling in DLBCL (Alizadeh et al., Nature 2000)

Ratio of hybridization of fluorescent experimental mRNA to fluorescent pooledreference mRNA

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

Differences Between GC-like and Activated B-like DLBCL

GC B-like DLBCL Activated B-like DLBCL

Cell of origin Germinal center B-cell Postgerminal center B-cell

Chromosomal alterations

t(14;18); gains of 12q Gains 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

Yes No

IHC Expression of CD10, BCL-6 Expression of MUM1

Clinical outcome Favorable (60% 5-year survival)

Poor (35% 5-year survival)

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

Immunohistochemistry (IHC) can be used to determine the GC B-like and Activated B-like

subtypes of DLBCL

NormalLymph Node

GC B-like DLBCL

Activated B-like/non-GC B-like DLBCL

C.P. Hans et al. Blood 2004, 103:275-282

CD10 BCL-6

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

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

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

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

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)

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

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

CHOP R-CHOP R-CHOP

The Hans Classifier – Controversies in the CHOP and R-CHOP Era

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

Survival association present

Survival association absent

Transcriptional Profile of DLBCL Subsequently Treated with R-CHOP

J.P. Jais et al. Leukemia 2008;22:1917-24

The recognition of GCB and ABC DLBCL is highly reproducible by gene expression profiling between

different centers

GCB Unclass ABC

GCB 53 3 0

Unclass 3 16 2

ABC 0 3 48

Center A

Center B

Concordant results in 117/128 (91%)

Unpublished data from Elias Campo, MD

Prognostic Impact of Gene Alterations in DLBCL in the Era of Rituximab

• MYC gene rearrangements

• CDKN2A (p16INK4/p14ARF) and CDKN2B (p15INK4) deletions

• p53 aberrations

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

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

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

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

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

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

p16 p14 p15

Gray: heterozygous deletionBlack: homozygous deletion

CDKN2A/B

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

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

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

Journal Club

4/10/12

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

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

cDNA Microarray - Methodology

17,856-GENE ARRAYLYMPHOCHIP

17,856-GENE ARRAYLYMPHOCHIP

Experimental sample

Reference poolof 9 differentlymphoma celllines

The fluorescence ratio was quantified for each gene and reflected the relativeabundance of the gene in eachexperimental mRNA sample compared to the reference pool

Experimental sample

Reference poolof 9 differentlymphoma celllines

Figure 1dendrogram

Each row: separate cDNAclone on the microarray

Each column: separate experimental mRNAsample

Results: ratio of hybridization of fluorescent experimental mRNA to fluorescent pooledreference mRNA

Figure 2

“Lymph node survivalsignature” 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

Figure 3

Figure 4

Figure 5