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Gene expression profiling in large cell lymphoma: emerging clinical applications

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Title: Gene expression profiling in large cell lymphoma: emerging clinical applications


1
Gene expression profiling in large cell lymphoma
emerging clinical applications
  • Timothy S. Fenske, M.D.
  • April 15, 2005


2
Diffuse Large B-cell Lymphoma
  • Most common NHL (1/3 of all NHL worldwide)
  • Heterogeneous morphology
  • Centroblastic type (80 of cases)
  • Immunoblastic (10 of cases)
  • Anaplastic and Plasmablastic variants also
  • Other subtypes recognized
  • Intravascular lymphoma
  • T cell (or histiocyte) rich B cell lymphoma
  • Lymphomatoid granulomatosis-type large BCL
  • Primary effusion lymphoma

3
DLBCL - pathogenesis
  • Rare for DLBCL to have unmutated Ig genes
  • Suggests they arise from cells that are GC or
    post-GC
  • Heterogeneous cytogenetic abnormalities
  • Translocations involving bcl-6 gene (3q27) are
    seen in 35 of cases
  • block the downregulation of bcl-6 that normally
    occurs during differentiation into post-germinal
    center cells.
  • Leads to repression of genes that induce
    cell-cycle arrest, apoptosis and differentiation.
  • Translocations involving bcl-2 gene, such as
    t(1418), seen in lt30
  • These cases may evolve from follicular lymphoma
  • Can see overexpression of bcl-2 even without
    translocations. Correlated with poor prognosis
    (indep of IPI) in some studies.

4
DLBCL clinical behavior
  • Wide variation
  • Most common presentation is rapidly enlarging
    lymphadenopathy
  • However, up to 40 can present with extranodal
    sites (such as GI, CNS, bone, skin)
  • Can see unusual presentations such as in
    intravascular, primary effusion
  • 25 stg I, 25 stg II, 50 stg III/IV
  • gt10cm masses in ¼ to 1/3 of patients
  • BM involved in approx 15

5
DLBCL prognosis highly variable
  • Overall about 40-50 have prolonged survival
  • International Prognostic Index
  • Age ( 60 vs gt60)
  • LDH (normal vs elevated)
  • PS (0-1 vs 2-4)
  • Stage (I/II vs III/IV)
  • Extranodal involvement (0-1 sites vs gt1 site)
  • Low (0-1 factors, 35 of pts) 73 5 yr OS
  • Low Intermed (2 factors, 27 of pts) 51
  • High Intermed (3 factors, 22 of pts) 43
  • High (4-5 factors, 16 of pts) 26

6
OS by IPI risk group
NEJM 1993
However, despite this, risk stratified therapies
for high risk patients have not emerged based
on IPI score (or other adverse features).
7
Hetergeneity at a molecular level also
  • Staudt et al were able to classify DLBCLs into 2
    different categories using DNA microarrays
  • Germinal Center B-cell like (GCB)
  • and
  • Activated B-cell like (ABC)

Nature, 2000
8
CD138
MUM1
bcl-6
CD10
large non-cleaved
small cleaved
MZL
DLBCL (ABC type)
(GC type)
B-CLL
Somatic hypermutation of Ig genes
9
Hetergeneity at a molecular level also
  • Some of the GCB genes were already known to be
    present on GC B-cells by immunohistochem CD10,
    CD38, bcl-6
  • Bcl-6 levels did not correlate with presence of
    translocation, suggesting that it was more
    related to GC phenotype and not translocation
  • ABC genes of interest
  • Bcl-2 (again level did not correlate with
    translocation)
  • MUM1 previously known to be translocated to Ig
    locus in MM cases
  • transiently induced during normal lymphocyte
    activation and is critical for the proliferation
    of B lymphocytes in response to signals from the
    antigen receptor

10
Molecular subtype correlated with survival
(No validation set, small number of tumors)
Nature, 2000
11
Molecular subtype can predict survival
  • Rosenwald et al, took 240 DLBCL specimens,
    identified GCB, ABC and Type 3 groups

NEJM, 2002
12
Again GCB subtype correlated with better survival
NEJM, 2002
13
Can a simplified microarray score predict
survival?
  • Rosenwald et al took their 160 of their 240 DLBCL
    specimens, and identified 17 genes that appeared
    particularly predictive of survival
  • Specifically chose genes from different
    biological pathways
  • GCB, MHC class II, lymph node groups
    (favorable)
  • proliferation, BMP-6, groups (unfavorable)
  • expression of each of the 17 genes then
    incorporated into a score

14
microarrays predict survival
NEJM, 2002
15
Microarray predicts survival independent of IPI
score
NEJM, 2002
16
Shipp et al could not predict based on cell of
origin
Later, Staudts group was able to resolve this
using their platform and probesets on these
samples
17
Moving toward a logistically easier assay
  • R. Levys group (Stanford) identified 36 genes
    from the literature to test in a qRT-PCR assay to
    predict survival in a group of 66 patients

NEJM, 2004
18
NEJM, 2004
19
NEJM, 2004
20
Can DLBCL be classfied into GC vs non-GC using
IHC instead?
  • Gene array is expensive and not logistically
    feasible for real-time processing of clinical
    specimens
  • qRT-PCR in theory could have a quick turn around
    but may not if samples need to be batched, etc.
  • Technical issues of appropriate controls,
    normalization to housekeeping gene
  • May be possible to classify cell of origin of
    DLBCL using limited number of IHC markers
  • Hans et al took 152 DLBCL cases, 142 of which had
    already been subjected to gene expression
    profiling, and made tissue microarrays
  • Subjected the tissue microarrays to staining
    using various antibodies
  • Were able to classify specimens as GC vs non-GC
    using 3 antibodies

21
CD138
MUM1
bcl-6
CD10
large non-cleaved
small cleaved
MZL
DLBCL (ABC type)
B-CLL
(GC type)
Somatic hypermutation of Ig genes
22
CD138
MUM1
bcl-6
CD10
large non-cleaved
small cleaved
MZL
MZL
DLBCL (ABC type)
(GC type)
B-CLL
(GC type)
B-CLL
23
IHC using 4 markers mirrors gene array data
Blood, 2004
24
IHC may be even better than gene array
Blood, 2004
These behaved more like non-GCB
IHC allows for direct evaluation of neoplastic
cells. Gene array and RT/PCR analyze cDNA from
the entire biopsy.
25
No obvious clinical differences for GC vs non-GC
26
Other markers may provide additional prognostic
value
Cyclin D2 only in non-GC cases. Esp poor
prognosis.
Bcl-2 not predictive on its own, but identifies
poor risk group of non-GC cases.
Other markers that may predict survival FoxP1
27
Confirmation from other groups
GC
IPIgt2, bcl-2, and non-GC
28
Rare subtypes of DLBCL
  • Primary cutaneous DLBCL of the leg
  • Primary breast DLBCL
  • Both have relatively poor prognoses
  • Both have recently been shown to be non-GC or
    ABC subtypes

29
Limitations of the data
  • Other factors besides cell of origin will
    impact prognosis / response to therapy
  • Liver and kidney function
  • Enzymes that may affect chemoRx metabolism or
    concentration in tumor cells
  • Enzymes involved in DNA repair
  • Surrounding stromal cells
  • Tumor volume / burden
  • Presumably why IPI predicts independent of cell
    of origin
  • The patients in these studies were treated with
    different regimens
  • Identification of molecular subgroups may
    identify treatments that are particularly
    effective for certain patients (e.g. Rituxan for
    bcl-2 DLBCL)

30
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31
ABC
ABC
GC
32
Summary
  • Gene array studies have led to a molecular scheme
    to classify DLBCL by cell of origin
  • This scheme has prognostic value, beyond that
    provided by strictly clinical parameters such as
    IPI score
  • This classification can be achieved with a
    limited number of IHC stains, without the need
    for extensive gene array analysis
  • Similar classification can be achieved using
    RT-PCR
  • Specific cell signalling pathways are being
    identified in molecular subtypes of DLBCL

33
Future Directions
  • Need to prospectively obtain gene array data from
    groups of patients treated with the same regimens
  • CALGB 50303 (pending) R-CHOP vs DA R-EPOCH
  • Gene array studies will be done on all patients
  • Prospective validation of GC vs non-GC
    classification using IHC
  • ? incorporation of additional markers such as
    cyclin D2
  • Testing of intensified therapies in a prospective
    manner in high risk patients
  • Identification of additional specific pathways /
    targets in molecular subtypes of DLBCL
  • e.g. NF-kB inhibition strategies in non-GC /
    ABC DLBCL

34
Blood, 2004
CD10 membrane-associated neutral endopeptidase
expressed in several tissues. In LN, expression
limited to GCs. Bcl-6 zinc finger
transcriptional repressor. Expressed in GCs and
a subset of T cells. MUM1 lymphoid specific
member of the IFN regulatory factor of TFs.
Normally expressed in plasma cells and a small
subset of GC cells.
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