Download Biological Prognostic Markers in Diffuse Large B

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Transcript
New prognostic markers that stratify
patients according to risk factors are
needed to provide the basis for
individually tailored treatment in
diffuse large B-cell lymphoma.
George Van Hook. Calm Boats. Oil on linen, 12˝ × 16˝.
Biological Prognostic Markers in Diffuse Large B-Cell Lymphoma
Anamarija M. Perry, MD, Zdravko Mitrovic, MD, and Wing C. Chan, MD
Background: Multiple novel therapeutic options have emerged in the treatment of non-Hodgkin lymphoma,
including monoclonal antibodies and different classes of biological agents. With this increased diagnostic
sophistication, novel prognostic markers are needed to stratify patients according to risk factors, particularly
those with a mechanistic underpinning, to provide the basis for individually tailored treatment.
Methods: Numerous prognostic markers have been proposed in patients with diffuse large B-cell lymphoma
(DLBCL), and this review discusses the more studied and the most widely used prognostic markers in DLBCL
in the rituximab era.
Results: Prognostic markers in DLBCL include a range of biomarkers assessed by morphology, immunohistochemistry, and relatively novel molecular methods including gene expression profiling, high-resolution array
comparative genomic hybridization, and next-generation sequencing. Most of these methods are not routinely
used due to substantial cost, technical complexity, and the requirement for fresh or frozen tissue.
Conclusions: Efforts are underway to translate previous microarray findings to platforms that can be readily
used in routine clinical practice with high reproducibility, precise measurements, and minimal loss of information.
At the present time, there is no consensus on which biological prognostic markers should be routinely assessed in
patients with DLBCL, and practices vary widely among different institutions. With more global approaches, the
ability to assess biomarkers in the cellular or tumor context may be possible, resulting in a better understanding
of their biological and prognostic significance.
Introduction
Non-Hodgkin lymphoma (NHL) is composed of a heterogeneous group of tumors arising from B or T/NK cells
at various stages of differentiation.1 In the last decade,
From the Department of Pathology and Microbiology at the
Nebraska Medical Center, Omaha, Nebraska (AMP, WCC), and
the Department of Internal Medicine, Clinical Hospital Dubrava,
Zagreb, Croatia (ZM).
Submitted January 13, 2012; accepted April 30, 2012.
Address correspondence to Wing C. Chan, MD, Department of
Pathology and Microbiology, 983135 Nebraska Medical Center,
Omaha, NE 68198-3135. E-mail: [email protected]
No significant relationship exists between the authors and the
companies/organizations whose products or services may be referenced in this article.
214 Cancer Control
clinical and laboratory investigations complemented
by novel molecular methods such as gene expression
profiling (GEP) and other genome-wide investigations
have helped to expand our understanding of the biology and diversity of different types of NHL. This is
reflected in an increase in the number of entities in the
recent World Health Organization (WHO) classification.
The guiding principle of the WHO classification is an
attempt to define “real” diseases that can be recognized
by pathologists using all available information: morphology, immunophenotype, genetic characteristics,
and clinical features.1 Multiple novel therapeutic options have emerged in the treatment of NHL, including
monoclonal antibodies and different classes of biological
agents. Therefore, due to increased diagnostic sophisJuly 2012, Vol. 19, No. 3
tication and therapeutic armamentarium, novel prognostic markers are needed to stratify patients according
to risk factors, particularly those with a mechanistic
underpinning, to provide the basis for individually tailored treatment.
Prognostic markers in NHL encompass not only a
wide range of traditional as well as novel biomarkers
assessed by different methods, but also “pure” clinical
markers represented by the International Prognostic
Index (IPI) and its variants. The most extensively studied
markers in NHL are pathological markers. Immunohistochemistry (IHC) is a commonly used method to separate
B- from T-cell lymphomas and to further classify different entities that may have prognostic significance. IHC
staining is widely available in routine clinical practice, is
easy to use, and is relatively inexpensive. However, the
differences in tissue processing, antibody clones used,
different staining protocols, and interobserver variability
contribute to poor reproducibility of IHC among different institutions.2 At the other end of the diagnostic spectrum are relatively novel molecular methods such as GEP,
high-resolution array comparative genomic hybridization
(aCGH), and next-generation genome sequencing. GEP
has revealed novel, biologically distinct subtypes within
NHL entities, providing the biological basis for targeted
treatment. However, these methods are not widely available due to substantial cost, technical complexity, and
the requirement of fresh or frozen tissue.
Diffuse large B-cell lymphoma (DLBCL) constitutes
30% to 40% of all NHL cases in Western countries and
represents a biologically heterogeneous group of tumors.1 One of the most important clinical predictors of
survival in DLBCL patients is the IPI, which uses patient
age, Ann Arbor tumor stage, serum lactate dehydrogenase, performance status, and number of involved
extranodal sites to identify patients as low risk, lowintermediate risk, high-intermediate risk, and high risk.3
Subsequent studies have validated the prognostic value
of the IPI in DLBCL patients.4,5 The addition of rituximab to the standard chemotherapy protocol of cyclophosphamide, doxorubicin, vincristine, and prednisone
(R-CHOP) has significantly improved the survival of
patients with DLBCL.6-10 Some authors have questioned
the validity of the IPI in the rituximab era and have proposed the revised IPI (R-IPI) for clinical stratification of
DLBCL patients. The R-IPI, compared to the “traditional”
IPI, distinguishes three separate prognostic groups —
very good, good, and poor — and allows for a simpler
and more accurate prediction model.11
Numerous prognostic markers have been proposed
in patients with DLBCL, and this review discusses the
more studied and the most widely used markers. Prognostic markers are applicable primarily to DLBCL, not
otherwise specified. They have not been sufficiently
studied in special entities of this disease and therefore
are not applicable to these entities. However, some speJuly 2012, Vol. 19, No. 3
cial entities of DLBCL, as defined by a variety of criteria,
have unique clinical implications and are also discussed.
GEP and Distinct Subgroups of DLBCL
GEP has defi ned at least three biologically and prognostically distinct subgroups of DLBCL: germinal center
B-cell–like (GCB) DLBCL, activated B-cell–like (ABC)
DLBCL, and primary mediastinal large B-cell lymphoma
(PMLBCL). The GCB subtype is believed to be derived
from germinal center B cells and maintains the GCB differentiation program, while the ABC subtype putatively
arises from B cells that are arrested in their differentiation toward plasma cells. Furthermore, survival was
significantly better for patients in the GCB subgroup
compared with those in the ABC subgroup.12-16
Since GEP requires fresh or frozen tissue and this
application is not widely available, multiple ontogenetic
biomarkers such as BCL6, GCET2 (HGAL), CD10, LMO2,
FOXP1, and PKC-β were tested by IHC and correlated
with survival.17 It is likely that at least part of the predictive power is related to their differential expression in
the GCB and ABC subtypes of DLBCL, although singly,
these markers are not sufficiently specific to classify the
two subtypes of DLBCL. Therefore, various immunohistochemical algorithms have been developed in paraffinembedded tissue to reproduce the GEP classification.18
The most widely used is the Hans algorithm, which uses
three markers (CD10, BCL6, and MUM1) to separate GCB
DLBCL from non–GCB DLBCL.19 A more recent Choi
algorithm, based on five immunohistochemical markers
(GCET1, CD10, BCL6, MUM1, and FOXP1), had concordance of 87% or higher with GEP results, which was
superior to the Hans algorithm.18,20 Meyer et al18 recently
re-examined a number of algorithms and also proposed
a new “Tally” method. They found that most of the published algorithms perform well, with > 80% concordance
with GEP-classified cases (Fig 1). Some reports used
differences in patient survival to assess the usefulness
of the classification algorithm; however, this is a flawed
approach as prognosis depends on many factors other
than classification, such as the size of the patient population, the characteristics of the different populations, and
how well or how uniformly treatment is administered.
The prognostic usefulness of DLBCL subtyping has
been questioned in the rituximab era. Some investigators reported no difference in survival between GCB and
ABC subtypes when rituximab was added to the chemotherapy regimen,21-23 while others have shown persistent
difference.10 All studies that showed no difference in
survival between the GCB and ABC subtypes were based
on IHC algorithms. A GEP study of 233 patients treated
with R-CHOP showed that patients with GCB DLBCL
still had significantly higher overall and progression-free
survival than patients with ABC DLBCL had.24
Bortezomib is a protease inhibitor that can inhibit
the NF-κB pathway by blocking IκBα degradation.25 Since
Cancer Control 215
ABC DLBCL has constitutively activated NF-κB pathway,13
a study by Dunleavy et al25 investigated whether the addition of bortezomib to doxorubicin-based chemotherapy
would preferentially improve survival of patients with
ABC DLBCL. They found that patients with the ABC
subtype, compared to those with the GCB subtype of
DLBCL, had significantly higher response and median
overall survival (OS) when bortezomib was combined
with chemotherapy. However, this is a relatively small
study of 49 patients with relapsed DLBCL, and the findings need to be confirmed in independent large cohorts
of de novo DLBCL patients.
Prognostic Models Based on
Gene Expression Signatures
A few prognostic models, based on the combination of
expressions of several genes, were proposed in patients
with DLBCL treated with rituximab. Malumbers et al26
proposed a quantitative real-time polymerase chain reaction (Q-RT-PCR)-based model for prediction of outcome
in DLBCL patients. The expression of six genes was
measured in paraffin-embedded tissues; LMO2, BCL6,
and FN1 were associated with longer survival, while
CCND2, SCYA3, and BCL2 were associated with shorter
survival. Alizadeh et al27 recently proposed a two-gene
model based on the expression of a tumor biomarker
LMO2 and a tumor microenvironment marker TNFRSF9
in patients with DLBCL. The two-gene model was an
independent predictor of survival in the multivariate
analysis. Since it seems unlikely that the entire biology of tumor cells and host/tumor interaction can be
captured by one transcript, efforts are underway to
translate previous microarray findings to a platform that
can be readily used in routine clinical practice with high
A
ABC
+
MUM1
(≥ 80%)
+
–
GCB
Choi algorithm
GCET1
(≥ 80%)
GCB
+
–
CD10
(≥ 30%)
+
+
ABC
FOXP1
(≥ 80%)
–
–
BCL6
(≥ 30%)
GCB
–
ABC
B
Tally algorithm
“+” = 1, “–” = 0
GCB
CD10 (+ or –)
GCET1 (+ or –)
ABC
Mum1 (+ or –)
FoxP1 (+ or –)
Score
GCB > ABC
or
Score (0, 1, 2)
Score (0, 1, 2)
ABC > GCB
If GCB Score = ABC SCore: LMO2 ≥ 30%
LMO2 < 30%
GCB
ABC
Fig 1. — Choi (A) and Tally (B) immunohistochemical algorithms. In the Tally algorithm, antibody results are not examined in a particular order. Two
antigens of germinal center B cells (GCB) and two antigens of activated B cells (ABC) are examined. The case is classified according to expression
of the higher number of GCB vs ABC-associated antigens. If an equal number of GCB-associated and ABC-associated antigens are positive, then
LMO2 determines the phenotype. (A) Reproduced with permission of American Association for Cancer Research from Choi WW, Weisenburger DD,
Greiner TC, et al. A new immunostain algorithm classifies diffuse large B-cell lymphoma into molecular subtypes with high accuracy. Clin Cancer Res.
2009;15(17):5494-5502, permission conveyed through Copyright Clearance Center, Inc. (B) Reproduced with permission. © 2011 American Society
of Clinical Oncology. All rights reserved. From Meyer PN, Fu K, Greiner TC, et al. Immunohistochemical methods for predicting cell of origin and
survival in patients with diffuse large B-cell lymphoma treated with rituximab. J Clin Oncol. 2011;29(2):200-207.
216 Cancer Control
July 2012, Vol. 19, No. 3
reproducibility and better quantitation than IHC assays
provide and with minimal loss of information.28,29
density (MVD) in DLBCL and found high MVD to be an
unfavorable prognostic factor.
MicroRNA Signature
Genomic Aberrations in DLBCL
MicroRNAs (miRNAs) have been associated with outcome of DLBCL patients in a number of studies. Roehle
et al30 described the global miRNA signature of B-cell
lymphomas, including 58 cases of DLBCL. Eight miRNAs were found to correlate with survival. Patients
with downregulated miR-21, miR-23A, miR-27A, and
miR-34A expression had an inferior OS, while patients
with low levels of miR-19A, miR-195, and miR-LET7G
had a shorter event-free survival (EFS). Patients with
low expression of miR-127 had low OS and EFS. Alencar et al31 studied expression of miRNAs in 176 DLBCL
samples from rituximab-treated patients and found that
increased expression of miR-18A was associated with
shorter OS. Increased expression of miR-181A was seen
in patients with longer EFS. In contrast, higher expression of miR-222 was associated with shorter EFS.
MiRNA expression can also distinguish GCB and
ABC subtypes of DLBCL. Several miRNAs, such as
miR-155, miR-21, and miR-221, were found to be more
highly expressed in the ABC subtype than in the GCB
subtype.32,33 Furthermore, high miR-21 expression was
associated with longer EFS in de novo DLBCL cases.32
Most of the reported series studies have been small
and different platforms have been used with inclusion
of variable number of miRNAs. The published fi ndings
will need to be validated, refined, and extended in additional investigations that address these issues.
A number of studies have investigated genomic aberrations in DLBCL and their influence on prognosis. Certain genetic aberrations occur at different frequencies
among DLBCL subtypes. The t(14;18) translocation and
amplification of 2p16 are associated with the GCB subtype, while trisomy 3 or gain/amplification of chromosome arm 3q is associated with the ABC subtype. The
ABC DLBCL subtype is further characterized by gain of
18q and loss of 6q.36
Scandurra et al37 analyzed samples from 124 rituximab-treated DLBCL patients using a high-density genome-wide single nucleotide polymorphism-based array.
They found 58 gains, 47 losses, 54 losses of heterozygosity, 5 recurrent amplifications, and 7 homozygous
deletions. Twenty recurrent genetic lesions showed an
impact on the clinical course, among which deletions
affecting the short arm of chromosome 8 — del(8p23.1),
del(8p), and del(8p23.1-21.2) — showed the strongest
association with the poor outcome. Lenz et al36 analyzed
203 DLBCL samples using high-resolution array comparative genomic hybridization (aCGH) and found two
recurrently altered minimal common regions restricted
to ABC DLBCL that predicted adverse survival: trisomy
3 and INK4A/ARF locus single/double deletion. Chigrinova et al38 characterized DLBCL with chromosome 7q
gain. The gain of 7q delineated a group of DLBCL with
distinct biological and clinical characteristics. Most of
the patients were females and had prolonged OS with
no bone marrow involvement and significantly lower involvement of extranodal sites. Salaverria et al39 recently
found that t(6;14)(p25;q32) translocation that deregulates IRF4 is associated with GCB subtype of DLBCL,
younger age at diagnosis, and a favorable outcome. It
is important to include a sufficient number of cases in
each category and perform multivariate analysis to reach
reliable conclusions.
Tumor Microenvironment
Recently, the tumor microenvironment has been shown
to be an important prognostic factor in patients with
DLBCL.24 Rosenwald et al13 identified four gene-expression signatures that predicted survival in CHOP-treated
DLBCL patients: GBC, lymph node, major histocompatibility complex (MHC) class II, and proliferation.
Lymph node signature reflected tumor microenvironment, which was recently subdivided into two components: stromal-1 signature and stromal-2 signature.24
High stromal-1 signature identifies tumors with vigorous extracellular-matrix deposition and infiltration by
monocytes/macrophages and predicts good prognosis,
while the stromal-2 signature largely reflects angiogenesis and blood vessel density in the tumor stroma, and
high expression portends poor prognosis. Meyer et al34
attempted to reproduce the stromal-1 signature using
an antibody against secreted protein, acidic and rich
in cysteine (SPARC) to evaluate its expression in the
tumor microenvironment. Patients with high SPARC
positivity in the tumor stroma had a significantly longer survival than those with low or no SPARC staining.
Cardesa-Salzmann et al35 recently attempted to simulate the stromal-2 signature by measuring microvessel
July 2012, Vol. 19, No. 3
Single Prognostic Biomarkers
TP53 and TP21
TP53 is a tumor suppressor gene that acts as a multifunctional transcription factor involved in cell cycle
arrest, apoptosis, cell differentiation, replication, DNA
repair, and maintenance of genomic stability. Mutations in TP53 have been described in 18% to 30% of
patients with DLBCL.17 Young et al40,41 identified TP53
mutations in 21% of DLBCL patients, and the OS was
significantly worse than that of patients with wild-type
TP53. Mutations in TP53 DNA-binding domains were
the strongest predictor of poor OS. Mutations in the
Loop-Sheet-Helix and Loop-L3 were associated with
significantly decreased OS, but OS was not significantly
affected by mutations in Loop-L2 (Fig 2).
Cancer Control 217
A
AA125
TP53 Exons
Number of
Mutations
AA186
AA224
AA261
AA306
4
5
6
7
8
9
1
33
17
30
21
0
L1
B
110 112
113
NH2
124 127
132 136
141 147
S1
S2
S3
123
S1
Loop-L1
p53 Protein
IL-6
S1
163
Loop-L2
Structure
LSH
287 279 274 264
COOH
258 251 250
S10
H2
164
H
181 177
194
L2
H1
195
Loop-L3
170
167
S2
237 236 233
S9
S8
216 214
207 204 199
S7
S6
L3
C
AA117-142
171-181
Conserved Regions
II
III
IV
V
Number of Mutations
11
12
28
16
D
234-258 270-286
12
11
R248
Frequency of Mutations
10
9
8
7
R175
R213
6
5
R273
R280
R282
K132
4
3
2
1
0
50
100
150
200
250
300
350
Amino Acid Sequence of p53 Protein
Fig 2. — Schematic representation of the TP53 gene and its mutations in diffuse large B-cell lymphoma. (A) The distribution of TP53 mutations in exons 4 to 9, (B) their relation to p53 protein structure, (C) the mutations in conserved regions, and (D) the distribution and frequency of TP53 mutations
with peaks at known hot spot exons depicted. This research was originally published in Blood. Young KH, Leroy K, Møller MB, et al. Structural profiles
of TP53 gene mutations predict clinical outcome in diffuse large B-cell lymphoma: an international collaborative study. Blood. 2008;112(8):30883098. © the American Society of Hematology.
218 Cancer Control
July 2012, Vol. 19, No. 3
The cyclin-dependent kinase inhibitor TP21 negatively regulates cell cycle progression and inhibits cellular proliferation. Although it is a downstream effector of
the TP53, its expression is controlled by TP53-dependent
and TP53-independent mechanisms.42 Multiple studies
have quantified expression of TP53 and TP21 by IHC.
Strong nuclear staining for TP53 without TP21 staining
has been associated with TP53 gene alterations and has
been used as an imperfect surrogate for mutated TP53
in some studies.42 When assessed by IHC staining alone,
TP53 has been shown to be an unreliable predictor of
survival. Some investigators found an association between high TP53 expression and adverse survival,43,44
while others failed to show this association.45,46 The
addition of TP21 to the IHC panel somewhat improved
the prognostic value of TP53 expression.42,47
lost expression has been unclear. MHCII gene expression is controlled by several transcription factors, including RFX, CREB, and NF-Y, which interact with a master
transactivator protein class II transactivator (CIITA) to
form an enhanceosome complex. Although overall infrequent, decreases in CIITA expression appear to be the
most prevalent mechanism of MHCII downregulation.50,51
In PMLBCL, CIITA translocation with a concomitant decrease in MHCII expression is frequently observed.52
Pasqualucci et al53 recently performed massive
parallel sequencing on DLBCL samples and found
frequent inactivating mutations and deletions in the
β2-microglobulin gene (B2M) (including homozygous
deletions, biallelic mutations, or a combination of these).
B2M encodes a polypeptide that associates with a 45kD heavy chain to form the MHC class I molecule on
the surface of all nucleated cells. Taken together, these
inactivating mutations and deletions predict the loss of
B2M, which is required for cell surface expression of
HLA class I molecules and may impair the recognition
of the tumor cells by cytotoxic T lymphocytes.
MHC Molecules
Loss of MHC class I and class II (HLA-DP and HLA-DR) expression has been reported to correlate with shortened
survival in patients with DLBCL.48,49 The mechanism for
A
B
1.0
1.0
BCL2 protein negative (n = 71)
BCL2 protein positive (n = 67)
0.6
0.4
BCL2 protein positive (n = 27)
0.8
Overall Survival
Overall Survival
0.8
BCL2 protein negative (n = 28)
0.6
0.4
0.2
0.2
P = .69
P = .095
0
2
4
6
8
0
10
2
C
4
1.0
10
BCL2 protein negative (n = 13)
BCL2 protein negative (n = 18)
BCL2 protein positive (n = 26)
(30% cutoff)
0.6
0.4
0.2
BCL2 protein positive (n = 31)
(10% cutoff)
0.8
Overall Survival
0.8
Overall Survival
8
D
1.0
0.6
0.4
0.2
P = .008
0
6
Time (years)
Time (years)
P = .002
2
4
6
Time (years)
8
10
0
2
4
6
8
10
Time (years)
Fig 3. — Correlation of BCL2 protein expression with overall survival in (A) diffuse large B-cell lymphoma (DLBCL) as a single entity, (B) germinal center
B-cell–like (GCB) subgroup, (C) activated B-cell–like (ABC) subgroup (30% cutoff), and (D) ABC subgroup (10% cutoff). BCL2 protein expression is
predictive of survival in the ABC subgroup only (C and D). Reproduced with permission. © 2006 American Society of Clinical Oncology. All rights
reserved. From Iqbal J, Neppalli VT, Wright G, et al. BCL2 expression is a prognostic marker for the activated B-cell–like type of diffuse large B-cell
lymphoma. J Clin Oncol. 2006;24(6):961-968.
July 2012, Vol. 19, No. 3
Cancer Control 219
BCL2
BCL2, an antiapoptotic protein, was originally discovered due to its involvement in the t(14;18)(q32;q21)
translocation, which juxtaposes the BCL2 gene (18q21)
to the immunoglobulin (Ig) heavy-chain locus enhancers and results in BCL2 overexpression. BCL2 protein is
overexpressed in approximately 47% to 58% of DLBCL
cases.17 BCL2 expression in the GCB subgroup of DLBCL
is mainly through the presence of the translocation.
However, BCL2 expression can also be upregulated by
alternative mechanisms such as NF-κB activation and
18q21 gain/amplification, as often observed in the ABC
subgroup of DLBCL, which lacks the t(14;18).54 Numerous studies have investigated the correlation between
BCL2 protein expression, BCL2 translocation, and outcome in patients with DLBCL with conflicting results.
Iqbal et al55 detected t(14;18)(q32;q21) translocation
in 17% of DLBCL cases based on fluorescence in situ
hybridization, and the great majority of cases were of
the GCB subtype. However, there was no significant
difference in survival between the t(14;18)-positive and
-negative patients in the GCB subgroup, in contrast to
the significantly poorer survival of ABC DLBCL with
high BCL2 expression, when treated with CHOP (Fig 3).
Data regarding the BCL2 protein expression and its
influence on survival in DLBCL patients are also controversial in the rituximab era. Some studies have found
that the addition of rituximab to standard chemotherapy
overcame the adverse prognostic influence of BCL2 expression.56,57 Others have shown that BCL2 expression
remained an adverse prognostic factor in the rituximab
era, primarily in the non–GCB subgroup of patients.54,58
Iqbal et al59 recently evaluated a series of R-CHOP treated
DLBCL patients who had GEP-defi ned DLBCL subsets
and found that BCL-2 expression is a significant predictor of survival in the GCB subgroup but not in the ABC
subgroup (Fig 4). The addition of rituximab appears
to have reduced the difference in survival between the
BCL2-positive and -negative groups in the ABC subset.
For GCB DLBCL, it may have improved the survival of
MYC and “Double-Hit” Lymphomas
MYC, located at chromosome band 8q24, encodes a transcription factor involved in the regulation of a variety of
cellular processes that include proliferation, cell cycle
control, metabolism, apoptosis, and cell migration.61
MYC is most commonly deregulated as a result of chromosomal translocation to an Ig gene locus in Burkitt lymphoma (BL), but MYC translocations also occur in 7% to
10% of DLBCL cases.62,63 A number of studies reported an
adverse prognostic impact of MYC on survival of patients
with DLBCL who were treated with rituximab. Rimsza et
al64 found that high level of MYC expression, assessed by
quantitative nuclease protection assay (qNPA) in paraffinembedded tissue, was an independent indicator of poor
survival. Several studies found that the presence of MYC
rearrangements by fluorescence in situ hybridization
(FISH) studies was an independent predictor of survival
in multivariate analysis.65-68 In addition, MYC translocation was reported to be especially predictive of survival
in the GCB subgroup of patients.65
B-cell lymphomas with concurrent IGH-BCL2 and
MYC rearrangements, called “double-hit lymphomas,”
are neoplasms with a spectrum of morphologic features
overlapping with BL, DLBCL, and B-cell lymphomas, unclassifiable, with features intermediate between DLBCL
and BL. These tumors, regardless of the histologic appearance, are characterized by aggressive clinical behavior, often complex karyotypes, and poor outcome.1,69-72
Double-hit DLBCL usually shows a high proliferative
index (average 80%) but lower than a typical BL, when
B
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0
0.9
OS
EFS
0.8
0.7
Proportion
Proportion
A
BCL2-negative patients to a significantly greater extent
than for the BCL2-positive subgroup. However, this
latter fi nding needs to be confi rmed as the OS does
not reach statistical significance in the multivariate
analysis. BCL2 mutations have been shown to occur
most frequently in the GCB subtype of DLBCL.60 It is
possible that some of the mutations that enhance the
anti-apoptotic function of BCL2 may be selected and
may add to the complexity of the analysis.
BCL2 protein expression
< 50%
≥ 50%
P = .009
0.6
0.5
0.4
0.3
0.2
n = 94
n = 74
0.1
P = .001
0.0
0
1
2
3
4
5
Years
6
7
8
9
10
11
0
1
2
3
4
5
6
7
8
9
10
11
Years
Fig 4. — Significant correlation of BCL2 protein level with overall survival (OS) and event-free survival (EFS) in GCB DLBCL. Reproduced with permission of American Association for Cancer Research from Iqbal J, Meyer PN, Smith LM, et al. BCL2 predicts survival in germinal center B-cell–like
diffuse large B-cell lymphoma treated with CHOP-like therapy and rituximab. Clin Cancer Res. 2011;17(24):7785-7795, permission conveyed through
Copyright Clearance Center, Inc.
220 Cancer Control
July 2012, Vol. 19, No. 3
assessed by IHC stain for Ki-67.70,72 The presence of
MYC translocation and expression level of MYC per se
may not be the best prognosticators. Other modifiers
of MYC activity, cooperative oncogenic pathways, and
MYC mutations have to be determined to provide a more
complete picture of MYC as a biomarker.
Ki-67
Ki-67 is a nuclear antigen expressed by cycling cells.
The percentage of Ki-67 expressing cells reflects the
proportion of the tumor cells that are actively cycling.17
The prognostic significance of Ki-67 expression in
DLBCL is controversial. Several studies conducted in
rituximab-treated patients showed that elevated Ki-67
expression was associated with inferior OS and EFS.73,74
However, the cutpoints used to defi ne “high” vs “low”
Ki-67 have differed among authors, thus making the
comparison of individual studies difficult. In the study
by Lenz et al,24 Ki-67 expression or the proliferative
index was not an independent predictor of survival in
rituximab-treated patients.
CD43
The CD43 molecule is a multifunctional type I transmembrane glycoprotein expressed in a variety of hematopoietic cells.75 The role of CD43 in B cells is not
completely clear, but coexpression of CD43 and CD20
on peripheral B cells is suggestive of malignancy.76 CD43
is expressed in 16% to 28% of DLBCL.77,78 Mitrovic et
al79 found that patients with CD43-positive DLBCL had
significantly lower complete response, OS, and EFS
compared with CD43-negative DLBCL patients. Interestingly, the effect of CD43 was significant in patients
treated with R-CHOP, while the significance was not
observed in the CHOP-treated cohort.
Special Entities of DLBCL
PMLBCL is a distinct subtype of DLBCL of putative thymic B-cell origin. Recent studies support the late germinal center or postgerminal center stage of differentiation. Most patients are in the third decade of life, with
a slight female predominance. The majority of patients
present at early stage of disease with mediastinal involvement, and bone marrow involvement is rare. Morphologically, tumor cells are characteristically associated
with compartmentalizing alveolar fibrosis. PMLBCL
expresses pan-B cell antigens and is positive for CD30
in the majority of cases. Tumor cells are also frequently
positive for IRF4/MUM1 and CD23 and are variably positive for BCL2 and BCL6.1,80 Several IHC markers were
proposed to aid in differentiating PMLBCL from other
types of DLBCL. These markers include, but are not
limited to, NFκB family member c-Rel, NFκB target gene
TRAF1, MAL antigen, dendritic cell marker TNFAIP2,
and a member of the TP53 family TP73L.1,81-84 PMLBCL
shows clonally rearranged Ig heavy- and light-chain
July 2012, Vol. 19, No. 3
genes, but most cases are surface Ig-negative. PMLBCL
shows a unique profile of chromosomal abnormalities including frequent gains of chromosomes 2p, 9p, 12q, Xq,
7q, and 9q, and losses involving 1p. Gains in 9p include
JAK2, PDL1, PDL2, and SMARCA2 genes,1,15,16,85 while
2p gains include the REL proto-oncogene. Interestingly,
PMLBCL shows a unique GEP, and it shares many expressed transcripts with classical Hodgkin lymphoma
cell lines and has been recently shown to have a high
frequency of translocations involving CIITA similar to
Hodgkin lymphoma.15,16,52 Patients with PMLBCL have a
similar survival to those with GCB DLBCL, with current
cure rates up to 80%.1
T-cell/histiocyte-rich large B-cell lymphoma
(THRLBCL) is a variant of DLBCL associated with a
prominent component of reactive T cells and also frequently histiocytes. The median patient age is the sixth
to seventh decade, with a slight male predominance. It
has been suggested that at least a proportion of cases
are pathogenetically related to, or derived from, nodular
lymphocyte predominant Hodgkin lymphoma. Compared to conventional DLBCL, THRLBCL more commonly presents with advanced-stage disease and bone marrow involvement. Morphologically, the large neoplastic
cells usually account for less than 10% of the cellular
population and are dispersed singly in a background
of small lymphocytes. Tumor cells express pan-B cell
markers and are usually negative for CD30 and CD15.
BCL2, BCL6, and EMA are variably expressed. The small
cells in the background are CD3-positive T cells of predominantly CD8-positive cytotoxic type. THRLBCL has
clonally rearranged Ig genes. BCL2 rearrangement is
present in approximately one-fourth of cases. THRLBCL
is often an aggressive lymphoma, with a 3-year OS rate
of 46%. Frequently advanced clinical stage at diagnosis
contributes to the aggressiveness of this lymphoma.
However, when matched for the IPI, THRLBCL and conventional DLBCL have similar outcomes.1,80,86
Intravascular large B-cell lymphoma (IVLBCL) is
a rare type of large B-cell lymphoma characterized by
selective growth of lymphoma cells within the lumina
of small blood vessels, particularly capillaries, but not
larger arteries and veins. This tumor is derived from
peripheral B cells, with the majority of cases showing
non–GCB phenotype. It occurs most commonly in the
sixth to seventh decade of life. Tumor cells lack the
expression of CD29 (β1 integrin) and CD54 (ICAM-1),
which are molecules important for transvascular lymphocyte migration. This might explain the propensity
of tumor cells to be localized inside the vessel lumens.
IVLBCL is a clinical mimicker of many diseases, and two
clinical variants are recognized: Western and Asian.
The Western form is most commonly characterized
by nonspecific, nonlocalizing neurologic symptoms or
skin lesions. However, any organ can be involved.1,80
The Asian variant, mostly reported by Japanese authors,
Cancer Control 221
is characterized by fever, hepatosplenomegaly, hemophagocytic syndrome with cytopenias, marrow involvement, and disseminated intravascular coagulation.1,80,87
IVLBCL expresses CD45 and pan-B cell markers. CD5,
CD10, or BCL6 is expressed in some cases, with about
20% frequency. Cytogenetic abnormalities involving
8p21, 19q13, 14q32, and chromosome 18 have been reported in the Asian variant. This tumor was invariably
fatal in the past, but more recent reports suggest that
aggressive chemotherapy can lead to complete remission and long-term survival in some patients. The Asian
variant has an aggressive clinical course, with a median
survival of 7 months.1,80
Epstein-Barr virus (EVB)–positive DLBCL of the
elderly is an EBV-associated clonal B-cell proliferation
occurring in patients older than 50 years without any
known immunodeficiency or prior lymphoma. It is postulated that this lymphoma results from immunologic
deterioration associated with aging. This entity has been
reported most commonly in Asians, with a frequency of
8% to 10% of all DLBCL cases among patients without
a documented predisposing immunodeficiency. Data
in the Western population are scarce, but the overall
incidence is about 3% in this patient population.88 The
median age of reported cases at diagnosis is 71 years,
with a slight male predominance. About 70% of patients
present with extranodal disease, with or without nodal
involvement, while 30% of patients have only nodal
disease. Morphologically, two subtypes are recognized:
polymorphic and large-cell lymphoma. Tumor cells
usually express pan-B cell markers, although they occasionally may lack CD20 expression. CD30 expression is
variable, and CD10 and BCL6 are usually negative, while
IRF4/MUM1 is commonly positive. The tumor cells
contain EBV, and EPV-encoded RNA (EBER) positivity
is demonstrated in the majority of tumor cells. Ig genes
are usually clonally rearranged. The clinical course is
aggressive, with a median survival of 2 years and a 5-year
survival rate of approximately 25%.1,80
De novo CD5-positive DLBCL is a subtype with CD5
expression. Most of the reports concerning this subtype are from Japan, where approximately 10% of all
de novo DLBCL cases express CD5. The median age
of patients is the seventh decade, with a slight female
predominance. Patients most commonly present in
higher clinical stages, and the majority have extranodal
involvement. The tumor cells are usually positive for
BCL2 and BCL6 and negative for CD10. CD23 and cyclin
D1 are negative. The majority of cases are classified immunophenotypically as non–GCB. BCL6 is rearranged
in 40% of cases. Described genetic aberrations include
gains of 10p14-15, 19q13, 11q21-24, and 16p and losses of
1q43-44 and 8p23. Compared with conventional DLBCL,
de novo CD5-positive DLBCL is associated with a more
aggressive clinical course, an overall worse prognosis,
and central nervous system (CNS) recurrence.1,89,90
222 Cancer Control
Other rare subtypes/variants of DLBCL with adverse
prognostic implications include plasmablastic lymphoma, ALK-positive large B-cell lymphoma, primary cutaneous DLBCL-leg type, DLBCL associated with chronic
inflammation, and primary effusion lymphoma.1
DLBCL in Immune-Privileged Sites:
CNS and Testis
Primary DLBCL of the CNS is relatively rare, representing < 1% of all NHL and approximately 2% to 3% of all
brain tumors. It occurs in both immunocompetent and
immunosuppressed individuals. Most immunocompetent patients are older, with a median age of 60 years
and a slight preponderance in males. Approximately
60% of all CNS DLBCL cases are located supratentorially, and multiple lesions are often present. Patients
most commonly present with focal neurological deficits. Most primary CNS DLBCL cases are of non–GCB
subtype and are usually negative for Epstein-Barr virus
when occurring in immunocompetent patients. The
most common genetic abnormality is BCL6 translocation
(30% to 40%). Commonly, there are deletions at 6q and
gains at 12q, 22q, and 18q21, with amplification of BCL2
and MALT1.1,91,92 The prognosis has been improved by
novel chemotherapeutic protocols that include methotrexate and high-dose cytarabine. The International
Extranodal Lymphoma Study Group (IELSG) reported
a complete remission rate of 46% in patients treated
with methotrexate and cytarabine compared with 18%
in patients treated with methotrexate alone. The 3-year
OS rates were 46% and 32% in the patients treated with
and without the addition of cytarabine, respectively.93
Most relapses occur in the CNS but can also involve
breast and testis.1
Primary DLBCL of the testis usually presents in
adults with median age in the sixth decade. The most
common clinical presentation is painless testicular enlargement with rapid progression. Local involvement
of the adjacent structures, as well as involvement of
the regional lymph nodes, can occur in the course of
disease. Most testicular DLBCL, like CNS types, are of
non–GCB subtype and have high proliferative activity.94
Genetic alterations in testicular DLBCL often comprise
complex abnormalities, including translocations, trisomies, amplifications, and deletions. The more common
alterations are abnormalities of 3q27 and 6q deletions.
Primary testicular DLBCL is an aggressive disease, with
frequent relapses and in general a poorer outcome than
that seen in “classic” DLBCL.95-97 Gundrum et al95 reported a median OS of 4.6 years, whereas the diseasespecific survival rates at 3, 5, and 15 years were 71.5%,
62.4%, and 43%, respectively.
Many cases of testicular and CNS DLBCL show decreased or no expression of HLA class I and II proteins,
thus allowing the tumor cells to escape immune attack. These tumors were found to have small deletions
July 2012, Vol. 19, No. 3
of 6p21.3 affecting the HLA region that contributes to
the loss of HLA class I and II proteins expression.1,98
Booman et al98 showed that loss of expression of HLADR at the mRNA level in testicular DLBCL is associated
with a significantly lower expression of many immuneregulated genes such as markers for T cells, NK cells,
macrophages, and antigen-presenting cells. The coordinate downregulation of these genes with HLA-DR levels
indicates a severe disruption of the immune response
in testicular DLBCL.
A summary of the different biological subtypes of
DLBCL and their impact on prognosis is provided in
the Table.
that the DLBCL developed from an occult small B-cell
lymphoma or that two unrelated lymphomas are present. Concordant bone marrow involvement has been
associated with the poorer outcome, while the data
regarding discordant involvement and its influence on
prognosis have been controversial. Sehn et al104 analyzed a series of 795 rituximab-treated DLBCL patients
and found that 67 (8.4%) had concordant and 58 (7.3%)
had discordant bone marrow involvement. The patients with concordant bone marrow involvement had
lower OS, while EFS was inferior in both concordant
and discordant involvement. In a multivariate analysis, concordant involvement remained an independent
predictor of EFS.
DLBCL in HIV Infection/AIDS
The association between HIV infection and the development of lymphoma has been observed since the early
phases of the AIDS epidemic. In 1986, the Centers for
Disease Control and Prevention recognized NHL as an
AIDS-defining illness. In the era prior to the introduction of highly active antiretroviral therapy (HAART),
NHL represented the second most frequent cancer associated with AIDS, after Kaposi sarcoma.99 DLBCL
is the most common type of AIDS-related lymphoma.
Following the introduction of HAART, the incidence
of HIV-related lymphomas has decreased, most prominently in primary CNS lymphoma. BL incidence also
decreased, with a relative increase in DLBCL.80,100,101 A
number of studies have shown improved survival in
AIDS-related NHL, including DLBCL, after the introduction of HAART therapy.99-102 Navarro et al103 found that
HIV-infected DLBCL patients treated with HAART and
chemotherapy had similar response rates to chemotherapy, OS, and EFS as HIV-negative DLBCL patients
receiving CHOP therapy.
Gray Zone Lymphomas
The 2008 WHO classification introduced two new entities in which features of DLBCL overlap with BL or with
classical Hodgkin lymphoma (CHL).1,105
B-cell lymphomas, unclassifiable, with features
intermediate between DLBCL and BL, are aggressive
lymphomas that have overlapping genetic, morphological, and IHC features of DLBCL and BL. These relatively
infrequent tumors usually present with widespread,
extranodal disease. Some cases resemble BL morphologically but have one or more immunophenotypic or
molecular genetic deviations that would exclude it from
the BL category. On the contrary, some cases have immunophenotypic and/or genetic features of BL but are
morphologically too atypical for BL. These cases tend
to have more complex genetic abnormalities than BL
has, and they are far more likely to have non–Ig-MYC
translocations. Some cases have concomitant BCL2
translocation (“double-hit” cases). B-cell lymphomas,
unclassifiable, with features intermediate between
DLBCL and BL, generally have an aggressive clinical
Bone Marrow Involvement in DLBCL
course and poor response to standard chemotherapy
Approximately 10% to 25% of DLBCL patients exhibit
regimens, with “double-hit” lymphomas having an esbone marrow involvement by lymphoma at the time
pecially poor prognosis.1,105
of diagnosis. Many have histologically concordant inB-cell lymphoma, unclassifiable, with features intervolvement with large B cells; however, 40% to 72% of
mediate between DLBCL and CHL, demonstrates overpatients have discordant marrow infi ltrates consisting
lapping clinical, morphological, and/or immunopheof mainly small B cells. In these cases, it is presumed
notypic features between CHL and DLBCL, especially
PMLBCL. These tumors usually occur
Table. — Different Biological Subtypes of Diffuse Large B-Cell Lymphoma and
in young men and present as a mediTheir Impact on Prognosis
astinal mass, with or without involvement of supraclavicular lymph nodes.
Good Prognosis
Intermediate Prognosis
Poor Prognosis
This diagnosis should be restricted to
DLBCL, GCB subtype
DLBCL, non-GCB subtype
IVLBCL
cases showing significant overlapping
PMLBCL
THRLBCL
EBV-positive DLBCL of the elderly
features with marked diagnostic discorCD5-positive DLBCL
Primary CNS DLBCL
dance between the morphology and the
Primary testicular DLBCL
immunophenotype. Recent evidence
from methylation analysis and genetic
CNS = central nervous system, DLBCL = diffuse large B-cell lymphoma, GCB = germinal
center B-cell–like, EBV = Epstein-Barr virus, IVLBCL = intravascular large B-cell lymphoma,
studies note that this group of cases
PMLBCL = primary mediastinal large B-cell lymphoma, THRLBCL = T-cell/histiocyte-rich
does have intermediate features belarge B-cell lymphoma.
tween typical CHL and PMLBCL.106,107
July 2012, Vol. 19, No. 3
Cancer Control 223
These cases also may relapse with more typical CHL
or PMLBCL compared to the original biopsy. These
lymphomas have an aggressive clinical course and a
poorer outcome than either PMLBCL or CHL has.1,105
There is currently no consensus on optimal treatment
of this entity, although some authors propose that CD20positive gray zone lymphomas should be treated with
immunochemotherapy with rituximab followed by radiation treatment.108
Conclusions
Numerous biological prognostic markers have been proposed in patients with diffuse large B-cell lymphoma
(DLBCL), and the significance of many of these that were
studied before the rituximab era need to be reassessed.
Prognostic markers are assayed by a variety of methods,
most commonly by morphology and immunohistochemistry. Although widely available and relatively cheap,
the immunohistochemistry method suffers from poor
reproducibility and difficulty in quantification due to
differences in tissue processing, staining protocols, and
interobserver variability. Molecular methods such as
gene expression profi ling (GEP), high-resolution array
comparative genomic hybridization, and next-generation
sequencing hold great promise in elucidating the pathogenesis and prognosis of DLBCL, but these methods are
not widely available due to substantial cost, technical
complexity, and requirement for fresh and frozen tissue. However, more focused assays can be designed
for further studies, and the ability to apply these assays
to formalin-fi xed, paraffin-embedded tissue would allow the inclusion of large patient cohorts to improve
statistical power.
Next-generation sequencing has detected numerous
mutations in patients with DLBCL, but whether any of
those mutations are important for prognosis, alone or in
combination, is not yet answered. However, such global
studies allow us to examine markers in the context of
other modifying factors and hence overcome the problems of single-marker studies. For example, BCL2 may
be an important biomarker, but its clinical significance
is influenced by other factors such as other biological
activities of the pathway that leads to its expression,
the coexisting factors such as MYC translocation, and
possibly even mutations that may affect its biological
activities. Thus, studying BCL2 expression alone as a
biomarker may not generate reproducible results from
different populations. A more global approach may allow all of these factors to be included in the analysis,
thereby producing a more meaningful biomarker profile.
At the present time, there is no consensus on which
biological prognostic markers should be routinely assessed in patients with DLBCL, and practices vary
widely among different institutions. With more global
approaches, such as those noted above, the ability to
assess biomarkers in the cellular or tumor context may
224 Cancer Control
be possible, resulting in a better understanding of their
biological and prognostic significance.
References
1. Swerdlow SH, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon,
France: IARC; 2008.
2. de Jong D, Rosenwald A, Chhanabhai M, et al. Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation
of tissue microarray as a prerequisite for broad clinical applications. A
study from the Lunenburg Lymphoma Biomarker Consortium. Clin Oncol.
2007;25(7):805-812.
3. A predictive model for aggressive non-Hodgkin’s lymphoma. The
International Non-Hodgkin’s Lymphoma Prognostic Factors Project. N
Engl J Med. 1993;329(14):987-994.
4. Nicolaides C, Fountzilas G, Zoumbos N, et al. Diffuse large cell
lymphomas: identification of prognostic factors and validation of the International Non-Hodgkin’s Lymphoma Prognostic Index. A Hellenic Cooperative Oncology Group Study. Oncology. 1998;55(5):405-415.
5. Wilder RB, Rodriguez MA, Medeiros LJ, et al. International prognostic index-based outcomes for diffuse large B-cell lymphomas. Cancer.
2002;94(12):3083-3088.
6. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus
rituximab compared with CHOP alone in elderly patients with diffuse largeB-cell lymphoma. N Engl J Med. 2002;346(4):235-242.
7. Sehn LH, Donaldson J, Chhanabhai M, et al. Introduction of
combined CHOP plus rituximab therapy dramatically improved outcome
of diffuse large B-cell lymphoma in British Columbia. J Clin Oncol.
2005;23(22):5027-5033.
8. Feugier P, Van Hoof A, Sebban C, et al. Long-term results of the
R-CHOP study in the treatment of elderly patients with diffuse large B-cell
lymphoma: a study by the Groupe d’Etude des Lymphomes de l’Adulte. J
Clin Oncol. 2005;23(18):4117-4126.
9. Habermann TM, Weller EA, Morrison VA, et al. Rituximab-CHOP
versus CHOP alone or with maintenance rituximab in older patients with
diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(19):3121-3127.
10. Fu K, Weisenburger DD, Choi WW, et al. Addition of rituximab to
standard chemotherapy improves the survival of both the germinal center
B-cell-like and non-germinal center B-cell-like subtypes of diffuse large
B-cell lymphoma. J Clin Oncol. 2008;26(28):4587-4594.
11. Sehn LH, Berry B, Chhanabhai M, et al. The revised International
Prognostic Index (R-IPI) is a better predictor of outcome than the standard
IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP.
Blood. 2007;109:1857-1861.
12. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse
large B-cell lymphoma identified by gene expression profiling. Nature.
2000;403(6769):503-511.
13. Rosenwald A, Wright G, Chan WC, et al. The use of molecular
profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002;346(25):1937-1947.
14. Wright G, Tan B, Rosenwald A, et al. A gene expression-based
method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A. 2003;100(17):9991-9996.
15. Rosenwald A, Wright G, Leroy K, et al. Molecular diagnosis of
primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma. J
Exp Med. 2003;198(6):851-862.
16. Savage KJ, Monti S, Kutok JL, et al. The molecular signature of
mediastinal large B-cell lymphoma differs from that of other diffuse large
B-cell lymphomas and shares features with classical Hodgkin lymphoma.
Blood. 2003;102(12):3871-3879.
17. Lossos IS, Morgensztern D. Prognostic biomarkers in diffuse large
B-cell lymphoma. J Clin Oncol. 2006;24(6):995-1007.
18. Meyer PN, Fu K, Greiner TC, et al. Immunohistochemical methods
for predicting cell of origin and survival in patients with diffuse large B-cell
lymphoma treated with rituximab. J Clin Oncol. 2011;29(2):200-207.
19. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the
molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1):275-282.
20. Choi WW, Weisenburger DD, Greiner TC, et al. A new immunostain
algorithm classifies diffuse large B-cell lymphoma into molecular subtypes
with high accuracy. Clin Cancer Res. 2009;15(17):5494-5502.
21. Nyman H, Adde M, Karjalainen-Lindsberg ML, et al. Prognostic
impact of immunohistochemically defined germinal center phenotype in
diffuse large B-cell lymphoma patients treated with immunochemotherapy.
Blood. 2007;109(11):4930-4935.
22. Ilić I, Mitrović Z, Aurer I, et al. Lack of prognostic significance of
the germinal-center phenotype in diffuse large B-cell lymphoma patients
treated with CHOP-like chemotherapy with and without rituximab. Int J
Hematol. 2009;90(1):74-80.
23. Seki R, Ohshima K, Fujisaki T, et al. Prognostic impact of immunoJuly 2012, Vol. 19, No. 3
histochemical biomarkers in diffuse large B-cell lymphoma in the rituximab
era. Cancer Sci. 2009;100(10):1842-1847.
24. Lenz G, Wright G, Dave SS, et al. Stromal gene signatures in largeB-cell lymphomas. N Engl J Med. 2008;359(22):2313-2323.
25. Dunleavy K, Pittaluga S, Czuczman MS, et al. Differential efficacy
of bortezomib plus chemotherapy within molecular subtypes of diffuse
large B-cell lymphoma. Blood. 2009;113(24):6069-6076.
26. Malumbres R, Chen J, Tibshirani R, et al. Paraffin-based 6-gene
model predicts outcome in diffuse large B-cell lymphoma patients treated
with R-CHOP. Blood. 2008;111(12):5509-5514.
27. Alizadeh AA, Gentles AJ, Alencar AJ, et al. Prediction of survival
in diffuse large B-cell lymphoma based on the expression of 2 genes reflecting tumor and microenvironment. Blood. 2011;118(5):1350-1358.
28. Payton JE, Grieselhuber NR, Chang LW, et al. High throughput
digital quantification of mRNA abundance in primary human acute myeloid
leukemia samples. J Clin Invest. 2009;119(6):1714-1726.
29. Williams PM, Li R, Johnson NA, et al. A novel method of amplification of FFPET-derived RNA enables accurate disease classification with
microarrays. J Mol Diagn. 2010;12(5):680-686.
30. Roehle A, Hoefig KP, Repsilber D, et al. MicroRNA signatures characterize diffuse large B-cell lymphomas and follicular lymphomas. Br J
Haematol. 2008;142(5):732-744.
31. Alencar AJ, Malumbres R, Kozloski GA, et al. MicroRNAs are independent predictors of outcome in diffuse large B-cell lymphoma patients
treated with R-CHOP. Clin Cancer Res. 2011;17(12):4125-4135.
32. Lawrie CH, Soneji S, Marafioti T, et al. MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma. Int J Cancer. 2007;121(5):1156-1161.
33. Jung I, Aguiar RC. MicroRNA-155 expression and outcome in diffuse large B-cell lymphoma. Br J Haematol. 2009;144(1):138-140.
34. Meyer PN, Fu K, Greiner T, et al. The stromal cell marker SPARC
predicts for survival in patients with diffuse large B-cell lymphoma treated
with rituximab. Am J Clin Pathol. 2011;135(1):54-61.
35. Cardesa-Salzmann TM, Colomo L, Gutierrez G, et al. High microvessel density determines a poor outcome in patients with diffuse large
B-cell lymphoma treated with rituximab plus chemotherapy. Haematologica. 2011;96(7):996-1001.
36. Lenz G, Wright GW, Emre NC, et al. Molecular subtypes of diffuse
large B-cell lymphoma arise by distinct genetic pathways. Proc Natl Acad
Sci U S A. 2008;105(36):13520-13525.
37. Scandurra M, Mian M, Greiner TC, et al. Genomic lesions associated with a different clinical outcome in diffuse large B-Cell lymphoma
treated with R-CHOP-21. Br J Haematol. 2010;151(3):221-231.
38. Chigrinova E, Mian M, Shen Y, et al. Integrated profiling of diffuse
large B-cell lymphoma with 7q gain. Br J Haematol. 2011;153(4):499-503.
39. Salaverria I, Philipp C, Oschlies I, et al. Translocations activating
IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults. Blood. 2011;118(1):139-147.
40. Young KH, Weisenburger DD, Dave BJ, et al. Mutations in the
DNA-binding codons of TP53, which are associated with decreased expression of TRAILreceptor-2, predict for poor survival in diffuse large Bcell lymphoma. Blood. 2007;110(13):4396-4405.
41. Young KH, Leroy K, Møller MB, et al. Structural profiles of TP53
gene mutations predict clinical outcome in diffuse large B-cell lymphoma:
an international collaborative study. Blood. 2008;112(8):3088-3098.
42. Winter JN, Li S, Aurora V, et al. Expression of p21 protein predicts
clinical outcome in DLBCL patients older than 60 years treated with R-CHOP
but not CHOP: a prospective ECOG and Southwest Oncology Group correlative study on E4494. Clin Cancer Res. 2010;16(8):2435-2442.
43. Zhang A, Ohshima K, Sato K, et al. Prognostic clinicopathologic
factors, including immunologic expression in diffuse large B-cell lymphomas. Pathol Int. 1999;49(12):1043-1052.
44. Ichikawa A, Kinoshita T, Watanabe T, et al. Mutations of the p53
gene as a prognostic factor in aggressive B-cell lymphoma. N Engl J Med.
1997;337(8):529-534.
45. Sohn SK, Jung JT, Kim DH, et al. Prognostic significance of bcl-2,
bax, and p53 expression in diffuse large B-cell lymphoma. Am J Hematol.
2003;73(2):101-107.
46. Maartense E, Kramer MH, le Cessie S, et al. Lack of prognostic significance of BCL2 and p53 protein overexpression in elderly patients with diffuse large B-cell non-Hodgkin’s lymphoma: results from a
population-based non-Hodgkin’s lymphoma registry. Leuk Lymphoma.
2004;45(1):101-107.
47. Visco C, Canal F, Parolini C, et al. The impact of P53 and P21(waf1)
expression on the survival of patients with the germinal center phenotype
of diffuse large B-cell lymphoma. Haematologica. 2006;91(5):687-690.
48. Miller TP, Lippman SM, Spier CM, et al. HLA-DR (Ia) immune phenotype predicts outcome for patients with diffuse large cell lymphoma. J
Clin Invest. 1988;82(1):370-372.
49. Rimsza LM, Roberts RA, Miller TP, et al. Loss of MHC class II
gene and protein expression in diffuse large B-cell lymphoma is related
to decreased tumor immunosurveillance and poor patient survival regardless of other prognostic factors: a follow-up study from the Leukemia and
July 2012, Vol. 19, No. 3
Lymphoma Molecular Profiling Project. Blood. 2004;103(11):4251-4258.
50. Cycon KA, Rimsza LM, Murphy SP. Alterations in CIITA constitute a common mechanism accounting for downregulation of MHC class
II expression in diffuse large B-cell lymphoma (DLBCL). Exp Hematol.
2009;37(2):184-194.
51. Rimsza LM, Chan WC, Gascoyne RD, et al. CIITA or RFX coding region loss of function mutations occur rarely in diffuse large B-cell
lymphoma cases and cell lines with low levels of major histocompatibility
complex class II expression. Haematologica. 2009;94(4):596-598.
52. Steidl C, Shah SP, Woolcock BW, et al. MHC class II transactivator CIITA is a recurrent gene fusion partner in lymphoid cancers. Nature.
2011;471(7338):377-381.
53. Pasqualucci L, Trifonov V, Fabbri G, et al. Analysis of the coding
genome of diffuse large B-cell lymphoma. Nat Genet. 2011;43(9):830-837.
54. Iqbal J, Neppalli VT, Wright G, et al. BCL2 expression is a prognostic marker for the activated B-cell-like type of diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(6):961-968.
55. Iqbal J, Sanger WG, Horsman DE, et al. BCL2 translocation defines a unique tumor subset within the germinal center B-cell-like diffuse
large B-cell lymphoma. Am J Pathol. 2004;165(1):159-166.
56. Mounier N, Briere J, Gisselbrecht C, et al. Rituximab plus CHOP
(R-CHOP) overcomes bcl-2-associated resistance to chemotherapy
in elderly patients with diffuse large B-cell lymphoma (DLBCL). Blood.
2003;101(11):4279-4284.
57. Wilson KS, Sehn LH, Berry B, et al. CHOP-R therapy overcomes
the adverse prognostic influence of BCL-2 expression in diffuse large Bcell lymphoma. Leuk Lymphoma. 2007;48(6):1102-1109.
58. Nyman H, Jerkeman M, Karjalainen-Lindsberg ML, et al. Bcl-2
but not FOXP1, is an adverse risk factor in immunochemotherapy-treated
non-germinal center diffuse large B-cell lymphomas. Eur J Haematol.
2009;82(5):364-372.
59. Iqbal J, Meyer PN, Smith LM, et al. BCL2 predicts survival in germinal center B-cell-like diffuse large B-cell lymphoma treated with CHOP-like
therapy and rituximab. Clin Cancer Res. 2011;17(24):7785-7795.
60. Schuetz JM, Johnson NA, Morin RD, et al. BCL2 expression in diffuse large B-cell lymphoma. Leukemia. 2011 Dec 22. Epub ahead of print.
61. Schrader A, Bentink S, Spang R, et al. High myc activity is an independent negative prognostic factor for diffuse large B cell lymphomas. Int
J Cancer. 2011 Sep 12. Epub ahead of print.
62. Dalla-Favera R, Bregni M, Erikson J, et al. Human c-myc onc gene
is located on the region of chromosome 8 that is translocated in Burkitt
lymphoma cells. Proc Natl Acad Sci U S A. 1982;79(24):7824-7827.
63. Kramer MH, Hermans J, Wijburg E, et al. Clinical relevance of
BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma.
Blood. 1998;92(9):3152-3162.
64. Rimsza LM, Leblanc ML, Unger JM, et al. Gene expression predicts overall survival in paraffin-embedded tissues of diffuse large B-cell
lymphoma treated with R-CHOP. Blood. 2008;112(8):3425-3433.
65. Yoon SO, Jeon YK, Paik JH, et al. MYC translocation and an increased copy number predict poor prognosis in adult diffuse large B-cell
lymphoma (DLBCL), especially in germinal centre-like B cell (GCB) type.
Histopathology. 2008;53(2):205-217.
66. Savage KJ, Johnson NA, Ben-Neriah S, et al. MYC gene rearrangements are associated with a poor prognosis in diffuse large Bcell lymphoma patients treated with R-CHOP chemotherapy. Blood.
2009;114(17):3533-3537.
67. Barrans S, Crouch S, Smith A, et al. Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma
treated in the era of rituximab. J Clin Oncol. 2010;28(20):3360-3365.
68. Zhang HW, Chen ZW, Li SH, et al. Clinical significance and prognosis of MYC translocation in diffuse large B-cell lymphoma. Hematol Oncol.
2011;29(4):185-189.
69. Niitsu N, Okamoto M, Miura I et al. Clinical features and prognosis
of de novo diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC
translocations. Leukemia. 2009;23(4):777-783.
70. Snuderl M, Kolman OK, Chen YB, et al. B-cell lymphomas with
concurrent IGH-BCL2 and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma
and diffuse large B-cell lymphoma. Am J Surg Pathol. 2010;34(3):327340.
71. Aukema SM, Siebert R, Schuuring E, et al. Double-hit B-cell lymphomas. Blood. 2011;117(8):2319-2331.
72. Li S, Lin P, Fayad LE, et al. B-cell lymphomas with MYC/8q24 rearrangements and [email protected]/t(14;18)(q32;q21): an aggressive disease
with heterogeneous histology, germinal center B-cell immunophenotype
and poor outcome. Mod Pathol. 2012;25(1):145-156.
73. Yoon DH, Choi DR, Ahn HJ, et al. Ki-67 expression as a prognostic
factor in diffuse large B-cell lymphoma patients treated with rituximab plus
CHOP. Eur J Haematol. 2010;85:149-157.
74. Broyde A, Boycov O, Strenov Y, et al. Role and prognostic significance of the Ki-67 index in non-Hodgkin’s lymphoma. Am J Hematol.
2009;84(6):338-343.
75. Remold-O’Donnell E, Zimmerman C, Kenney D, et al. Expression
Cancer Control 225
on blood cells of sialophorin, the surface glycoprotein that is defective in
Wiskott-Aldrich syndrome. Blood. 1987;70(1):104-109.
76. Knowles DM. Immunophenotypic markers useful in the diagnosis and classification of hematopoietic neoplasms. In: Knowles DM, ed.
Neoplastic Hematology. 2nd ed. Philadelphia, PA: Lippincott Williams &
Wilkins; 2001:93-226.
77. Lai R, Weiss LM, Chang KL, et al. Frequency of CD43 expression in non-Hodgkin lymphoma. A survey of 742 cases and further
characterization of rare CD43+ follicular lymphomas. Am J Clin Pathol.
1999;111(4):488-494.
78. Gelb AB, Rouse RV, Dorfman RF, et al. Detection of immunophenotypic abnormalities in paraffin-embedded B-lineage non-Hodgkin’s lymphomas. Am J Clin Pathol. 1994;102(6):825-834.
79. Mitrovic Z, Ilic I, Nola M, et al. CD43 expression is an adverse prognostic factor in diffuse large B-Cell lymphoma. Clin Lymphoma Myeloma.
2009;9(2):133-137.
80. Jaffe Es, Harris NL, Vardiman JW, et al, eds. Hematopathology.
Philadelphia, Pa: Elsevier Saunders; 2011.
81. Copie-Bergman C, Plonquet A, Alonso MA, et al. MAL expression in lymphoid cells: further evidence for MAL as a distinct molecular marker of primary mediastinal large B-cell lymphomas. Mod Pathol.
2002;15(11):1172-1180.
82. Zamò A, Malpeli G, Scarpa A, et al. Expression of TP73L is a helpful diagnostic marker of primary mediastinal large B-cell lymphomas. Mod
Pathol. 2005;18(11):1448-1453.
83. Rodig SJ, Savage KJ, LaCasce AS, et al. Expression of TRAF1
and nuclear c-Rel distinguishes primary mediastinal large cell lymphoma
from other types of diffuse large B-cell lymphoma. Am J Surg Pathol.
2007;31(1):106-112.
84. Kondratiev S, Duraisamy S, Unitt CL, et al. Aberrant expression
of the dendritic cell marker TNFAIP2 by the malignant cells of Hodgkin
lymphoma and primary mediastinal large B-cell lymphoma distinguishes
these tumor types from morphologically and phenotypically similar lymphomas. Am J Surg Pathol. 2011;35(10):1531-1539.
85. Rui L, Emre NC, Kruhlak MJ, et al. Cooperative epigenetic modulation by cancer amplicon genes. Cancer Cell. 2010;18(6):590-605.
86. Achten R, Verhoef G, Vanuytsel L, et al. T-cell/histiocyte-rich
large B-cell lymphoma: a distinct clinicopathologic entity. J Clin Oncol.
2002;20(5):1269-1277.
87. Murase T, Yamaguchi M, Suzuki R, et al. Intravascular large Bcell lymphoma (IVLBCL): a clinicopathologic study of 96 cases with special reference to the immunophenotypic heterogeneity of CD5. Blood.
2007;109(2):478-485.
88. Caponetti GC, Bhagavathi S, Torabi A, et al. EBV-driven diffuse
large B-cell lymphoma in the elderly: a diagnostic entity? Mod Pathol.
2010;23(suppl 1). Abstract 1295.
89. Yamaguchi M, Nakamura N, Suzuki R, et al. De novo CD5+ diffuse
large B-cell lymphoma: results of a detailed clinicopathological review in
120 patients. Haematologica. 2008;93(8):1195-1202.
90. Westin J, McLaughlin P. De novo CD5+ diffuse large B-cell lymphoma: a distinct subset with adverse features, poor failure-free survival and
outcome with conventional therapy. Leuk Lymphoma. 2010;51(1):161-163.
91. Hattab EM, Martin SE, Al-Khatib SM, et al. Most primary central
nervous system diffuse large B-cell lymphomas occurring in immunocompetent individuals belong to the nongerminal center subtype: a retrospective analysis of 31 cases. Mod Pathol. 2010;23(2):235-243.
92. Gualco G, Weiss LM, Barber GN, et al. Diffuse large B-cell lymphoma involving the central nervous system. Int J Surg Pathol. 2011;19(1):4450.
93. Ferreri AJ, Reni M, Foppoli M, et al. High-dose cytarabine plus
high-dose methotrexate versus high-dose methotrexate alone in patients with primary CNS lymphoma: a randomised phase 2 trial. Lancet.
2009;374(9700):1512-1520.
94. Booman M, Douwes J, Glas AM, et al. Primary testicular diffuse
large B-cell lymphomas have activated B-cell-like subtype characteristics.
J Pathol. 2006;210(2):163-171.
95. Gundrum JD, Mathiason MA, Moore DB. Primary testicular diffuse
large B-cell lymphoma: a population-based study on the incidence, natural history, and survival comparison with primary nodal counterpart before
and after the introduction of rituximab. J Clin Oncol. 2009;27(31):52275232.
96. Mazloom A, Fowler N, Medeiros LJ, et al. Outcome of patients with
diffuse large B-cell lymphoma of the testis by era of treatment: the MD
Anderson Cancer Center experience. Leuk Lymphoma. 2010;51(7):12171224.
97. Horne MJ, Adeniran AJ. Primary diffuse large B-cell lymphoma of
the testis. Arch Pathol Lab Med. 2011;135(10):1363-1367.
98. Booman M, Douwes J, Glas AM, et al. Mechanisms and effects of
loss of human leukocyte antigen class II expression in immune-privileged
site-associated B-cell lymphoma. Clin Cancer Res. 2006;12(9):26982705.
99. Tirelli U, Spina M, Gaidano G, et al. Epidemiological, biological
and clinical features of HIV-related lymphomas in the era of highly active
226 Cancer Control
antiretroviral therapy. AIDS. 2000;14(12):1675-1688.
100. Kirk O, Pedersen C, Cozzi-Lepri A, et al. Non-Hodgkin lymphoma
in HIV-infected patients in the era of highly active antiretroviral therapy.
Blood. 2001;98(12):3406-3412.
101. Hoffmann C, Wolf E, Fätkenheuer G, et al. Response to highly active antiretroviral therapy strongly predicts outcome in patients with AIDSrelated lymphoma. AIDS. 2003;17(10):1521-1529.
102. Mounier N, Spina M, Gabarre J, et al. AIDS-related non-Hodgkin
lymphoma: final analysis of 485 patients treated with risk-adapted intensive chemotherapy. Blood. 2006;107(10):3832-3840.
103. Navarro JT, Lloveras N, Ribera JM, et al. The prognosis of HIV-infected patients with diffuse large B-cell lymphoma treated with chemotherapy and highly active antiretroviral therapy is similar to that of HIV-negative
patients receiving chemotherapy. Haematologica. 2005;90(5):704-706.
104. Sehn LH, Scott DW, Chhanabhai M, et al. Impact of concordant
and discordant bone marrow involvement on outcome in diffuse large Bcell lymphoma treated with R-CHOP. J Clin Oncol. 2011;29(11):1452-1457.
105. Hasserjian RP, Ott G, Elenitoba-Johnson KS, et al. Commentary
on the WHO classification of tumors of lymphoid tissues (2008): “Gray
zone” lymphomas overlapping with Burkitt lymphoma or classical Hodgkin
lymphoma. J Hematop. 2009;2(2):77-81.
106. Eberle FC, Rodriguez-Canales J, Wei L, et al. Methylation profiling
of mediastinal gray zone lymphoma reveals a distinctive signature with elements shared by classical Hodgkin’s lymphoma and primary mediastinal
large B-cell lymphoma. Haematologica. 2011;96(4):558-566.
107. Eberle FC, Salaverria I, Steidl C, et al. Gray zone lymphoma: chromosomal aberrations with immunophenotypic and clinical correlations.
Mod Pathol. 2011;24(12):1586-1597.
108. Grant C, Dunleavy K, Eberle FC, et al. Primary mediastinal large Bcell lymphoma, classic Hodgkin lymphoma presenting in the mediastinum,
and mediastinal gray zone lymphoma: what is the oncologist to do? Curr
Hematol Malig Rep. 2011;6(3):157-163.
July 2012, Vol. 19, No. 3