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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. 8.6%. 11.7%. 2.5%. 33%. 6.0%. 6.7%. 9.4%. 22.1%.

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diagnostically and prognostically significant genetic alterations in diffuse large b cell lymphoma

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
Frequency of B-and T-Cell Lymphomas

8.6%

11.7%

2.5%

33%

6.0%

6.7%

9.4%

22.1%

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

normal b cell differentiation
Normal B-Cell Differentiation

NEJM 2000, 343;108-117

the germinal center
The Germinal Center

Lymph node

Germinal Center

NEJM 2000, 343;108-117

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

diffuse large b cell lymphoma
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 lymphoma1
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
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
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)
slide10
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

slide11
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
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 profiling1
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
DNA Microarray - Methodology

DLBCL1

DLBCL2

+

+

+

+

+

LYMPHOCHIP

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

dna microarray methodology1
DNA Microarray - Methodology

DLBCL1

DLBCL1

DLBCL2

DLBCL2

LYMPHOCHIP

LYMPHOCHIP

Both sets of DLBCL cDNA are labelled

with different fluorescent tags, in this

case red and green

Gene array lymphochip is incubated

with the tagged DLBCL cDNAs, which

bind to the matching genes printed

on the array

dna microarray methodology2
DNA Microarray - Methodology

DLBCL1

DLBCL2

LYMPHOCHIP

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.

gene expression profiling in dlbcl alizadeh et al nature 2000
Gene Expression Profiling in DLBCL (Alizadeh et al., Nature 2000)

Ratio of

hybridization of fluorescent

experimental mRNA to

fluorescent pooled

reference mRNA

gene expression profiling in dlbcl alizadeh et al nature 20001
Gene Expression Profiling in DLBCL (Alizadeh et al., Nature 2000)
  • 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
  • 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
  • Hierarchical clustering of the genes selectively expressed in GC B-like DLBCL and activated B-like DLBCL, which was determined from Fig 3b
slide20
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

slide21
Immunohistochemistry (IHC) can be used to determine the GC B-like and Activated B-like subtypes of DLBCL

Normal

Lymph Node

CD10

BCL-6

GC B-like DLBCL

Activated B-like/non-GC

B-like DLBCL

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

slide22

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
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
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
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

Human IgG1

constant region

B-cell

CD20

Human k constant

region

Murine antigen binding domain

effects of rituximab
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)
slide27
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

slide28

The overall survival rates for control and immunochemotherapy-treated DLBCL patients according to immunohistochemical and clinical factors

CHOP

R-CHOP

R-CHOP

H. Nyman et al. Blood 2007;109:4930-4935

the hans classifier controversies in the chop and r chop era
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

transcriptional profile of dlbcl subsequently treated with r chop
Transcriptional Profile of DLBCL Subsequently Treated with R-CHOP

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

slide31
The recognition of GCB and ABC DLBCL is highly reproducible by gene expression profiling between different centers

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
Prognostic Impact of Gene Alterations in DLBCL in the Era of Rituximab
  • MYC gene rearrangements
  • CDKN2A (p16INK4/p14ARF) and CDKN2B (p15INK4) deletions
  • p53 aberrations
slide33
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
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
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

slide36
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

slide37

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

slide38

Schematic representation of the 9p21 locus and its genomic alterations detected by a dedicated quantitative multiplex PCR of short fragments (QMPSF) assay in DLBCL.

CDKN2A/B

p16

p14

p15

Jardin F et al. Blood 2010;116:1092-1104

Gray: heterozygous deletion

Black: homozygous deletion

summary
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
summary1
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
summary2
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
aim of study
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
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
cDNA Microarray - Methodology

Experimental

sample

Experimental

sample

Reference pool

of 9 different

lymphoma cell

lines

Reference pool

of 9 different

lymphoma cell

lines

17,856-GENE ARRAY

LYMPHOCHIP

17,856-GENE ARRAY

LYMPHOCHIP

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

slide47

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

Figure 1

dendrogram

slide49

“Proliferation signature”, a

quantitative measurement of tumor

cell proliferation, confers an inferior

survival

“Germinal center B-cell signature”

with BCL-6 and CD10 expression

“Lymph node survival

signature”  host response to lymphoma, is associated with  survival times

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