Introduction
The amount of data we need to digest becomes almost too large: data from large sequencing
experiments and also data from the increasing number of scientist around the world.
Looking at the series of articles, I had to go through for this review I sometimes
feel overwhelmed. Nevertheless, I have tried to make my personal selection again.
Biology of lymphoma
The possibilities of generating large amounts of data are enormous indeed. The challenge
is to generate meaningful information from the results of large sequencing approaches.
A nice example is the approach by Cimmino et al. [1]. They focus on ‘ten-eleven translocation
1’ (TET1) which is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic
stem cells, and the loss of 5hmC in many tumours suggests a critical role for the
maintenance of this epigenetic modification. In mice, they found that deletion of
TET1 promoted the development of B cell non-Hodgkin lymphoma (B-NHL). TET1 appeared
to be required for maintenance of the normal abundance and distribution of 5hmC, which
prevented hypermethylation of DNA, and for regulation of the B cell lineage and of
genes encoding molecules involved in chromosome maintenance and DNA repair. Then,
they reasoned that based on these data, TET1-deficient B cell lymphomas might have
specific mutations. Whole-exome sequencing (WES) indeed confirmed that these mutations
were frequently found in B-NHL in which TET1 was hypermethylated and transcriptionally
silenced. They conclude that TET1 acts as a tumour suppressor of B-NHL. A more inventory
approach was chosen by Braggio et al. [2] who performed a genome-wide analysis of
19 primary diffuse large B cell lymphomas (DLBCLs) of the central nervous system (CNS)
by array comparative genomic hybridization (CGH) and WES. They found biallelic inactivation
of TOX and PRKCD, which is not present in systemic DLBCL. In addition, they found
a high prevalence of MYD88 mutations (79 %) and CDKN2A biallelic loss (60 %) and alterations
in many other genes of the NF-κB pathway, but these are not unique for DLBCL of the
CNS. Overall NF-κB pathways were altered in >90 % of these cases. These findings indicate
that primary DLBCL of the CNS genetically differs from DLBCL from other sites, but
that there are also commonly affected pathways. A similar approach (using large amounts
of data from multiple approaches on a rare tumour) was taken by Lee et al. [3] for
34 cases of extranodal NK/T cell lymphoma nasal type (ENKL). They performed multiple
types of next-generation sequencing, including WES (9 cancer tissues and 4 cancer
cell lines), targeted sequencing (21 cancer tissues) and RNA sequencing (3 cancer
tissues and 4 cancer cell lines). Mutations were found most frequently in three genes,
STAT3, BCOR and MLL2 (which were present in nine, seven and six cancer samples, respectively),
whereas there were only two cases of Janus kinase 3 (JAK3) mutation. In total, JAK/STAT
pathway- and histone modification-related genes accounted for 55 and 38 % of the cancer
samples, respectively, and their involvement in ENKL pathogenesis was also supported
by gene expression analysis. They conclude that they found several novel driver genes
of ENKL, which may be future therapeutic targets in this disease. Finally, with the
present approach to sequencing, also more data regarding so-called pseudogenes become
available and it has become clear that these may have more important roles than previously
thought. Karreth et al. [4] show that mice engineered to overexpress either the full-length
murine B-Raf pseudogene Braf-rs1 or its pseudo ‘CDS’ or ‘3′ UTR’ develop an aggressive
malignancy resembling human DLBCL. They show that Braf-rs1 and its human ortholog,
BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous
RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo.
Furthermore, they show that transcriptional or genomic aberrations of BRAFP1 occur
frequently in multiple human cancers, including B cell lymphomas. These data indicate
that indeed also pseudogenes are relevant players in the development of B cell lymphomas.
All in all, these studies show that we seem to be only at the beginning of understanding
the complexity of the molecular basis of cancer and specifically lymphomas.
B cell lymphomas
One of the important drivers of B cell lymphomas, especially low-grade types, is the
signalling though the antigen receptor. Many studies have addressed the composition
of the immunoglobulin genes to get a better grip on the understanding of its role.
Berget et al. [5] investigated the V family usage in 99 cases of low-grade follicular
lymphoma (FL) and correlated the results with outcome. They found preferred usage
of immunoglobulin heavy variable 3 (IGHV3) (58 %), and those patients with IGHV5 or
more than one V family usage had a significantly worse outcome. In contrast with chronic
lymphatic leukaemia (CLL), there was no difference in the prognosis between patients
who had unmutated versus mutated sequences. These results indicate that the specific
antigen receptor is probably quite relevant, but that more detailed understanding
of the antigen (if any) involved is urgently needed. An example of a much more detailed
approach is the work by Green et al. [6], who analyzed purified FL cells and identified
additionally recurrently mutated genes and confirmed mutations of one or more chromatin
modifier genes in almost all cases. They defined the hierarchy of somatic mutations
arising during tumour evolution by analyzing the phylogenetic relationship of somatic
mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients.
CREBBP mutations were most significantly enriched within the earliest progenitor cell.
These mutations were associated with a signature of decreased antigen presentation
characterized by reduced transcript and protein abundance of MHC class II on tumour
B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent
transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation
of T cells in vitro compared with wild-type B cells from the same tumour. Transcriptional
signatures of tumour-infiltrating T cells were indicative of reduced proliferation,
and this corresponded to decreased frequencies of tumour-infiltrating CD4 helper T
cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP
mutation as an early event in FL evolution that contributes to immune evasion via
decreased antigen presentation.
Further proof of the role of antigens as a driver of B cell lymphoma, in this case
mantle cell lymphoma (MCL), comes from Xochelli et al. [7]. They found the AID full-length
transcript and the most frequent splice variants (AID-ΔE4a, AID-ΔE) in 128 (96 %),
96 (72 %) and 130 cases (98 %) of MCL, respectively. Higher AID full-length transcript
levels were significantly associated with lack of somatic hypermutation within the
IGHV genes. Although most cases exhibited low levels of intraclonal diversification,
analysis of the mutational activity revealed precise targeting of somatic hypermutation
indicative of an active, ongoing interaction with antigen(s). These findings indeed
strongly allude to antigen involvement in the natural history of MCL.
Pan et al. [8] hypothesized that epigenetic changes are important in the progression
of DLBCL. They compared the genome-wide methylation profile of relapsed DLBCL with
that of the original tumour and found a ‘relapse-associated methylation signature’
that included the transforming growth factor beta (TGF-β) receptor pathway. They also
observed decreased intra-tumour methylation heterogeneity from diagnosis to relapsed
tumour samples. Relapse-free patients display lower intra-tumour methylation heterogeneity
at diagnosis compared with relapsed patients in an independent validation cohort.
A better understanding of the underlying mechanism may therefore be of help to get
a better idea on tumour progression in DLBCL.
A more targeted approach was taken by Dubois et al. [9] with a focus on enhancer of
zeste homolog 2 (EZH2), of which recurrent somatic heterozygous gain-of-function mutations
of EZH2 have been identified in DLBCL. Since EZH2 inhibitors are being tested in phase
1 and 2 clinical trials, they want to develop a biomarker that predicts effect. By
immunohistochemistry, they determined the methylation profiles of the DLBCL from 82
patients as well as the mutational profiles of EZH2 in the lymphomas of 32 patients
with DLBCL with a next-generation sequencing (NGS) approach (panel of 34 genes involved
in lymphomagenesis). They developed a score based on H3K27me2 and H3K27me3 expression
that distinguished patients with wild-type (WT) EZH2 and patients with EZH2 Y641 mutation.
NGS analysis revealed a subclonal EZH2 mutation pattern in EZH2 mutant patients with
WT-like immunohistochemistry (IHC) methylation profiles, while associated mutations
capable of upregulating EZH2 were detected in WT EZH2 patients with mutant-like IHC
methylation profiles. The next step is going to show that this score is really predicting
therapeutic response of EZH inhibitors.
T cell lymphoma
Cresenzo et al. [10] focus with their genomic evaluation on anaplastic lymphoma kinase
(ALK)-negative (ALK−) anaplastic large cell lymphoma (ALCL). They identified activating
mutations of JAK1 and/or STAT3 genes in approximately 20 % of 155 ALK− ALCLs and demonstrated
that 38 % of systemic ALK− ALCLs displayed double lesions. Recurrent chimeras combining
a transcription factor (NFkB2 or NCOR2) with a tyrosine kinase (ROS1 or TYK2) were
also discovered in WT JAK1/STAT3 ALK− ALCL. All these aberrations lead to the constitutive
activation of the JAK/STAT3 pathway, which was proved to be oncogenic. Finally, they
showed that JAK/STAT3 pathway inhibition impairs cell growth in vitro and in vivo,
and this is therefore a rationale for a new treatment approach.
Epidemiology of lymphoma
Normally, we deal with the diagnosis of lymphomas and have good ideas on the treatments
that will follow our diagnosis as well as the resulting outcomes. We even are aware
of secondary cancers, which is especially a burden in survivors of Hodgkin lymphoma
(HL). I think it is also relevant to have an idea of less visible consequences. Glimelius
et al. [11] analyzed data from a population-based registry from Sweden on almost 2000
HL patients, with clinical data of more than 1000 of them, and compared these with
those of a matched cohort. The data include patients diagnosed between 1992 and 2009
with follow-up until 2013. The results indicate that HL survivors have a 30–50 % increase
in the risk of work loss, even when this was adjusted for secondary effects like cardiovascular
disease and secondary cancers. This underpins the major effect a cancer diagnosis
has for patients. This is an area that may be subject of overdiagnosis, and thus,
major unnecessary impact is in paediatric cases, especially paediatric nodal marginal
zone B cell lymphoma (NMZL). Excellent work from Kluin et al. [12] showed that the
spectrum of paediatric marginal zone hyperplasia to lymphoma may have a common cause—a
Haemophilus influenzae-driven immune response. In six children with polyclonal marginal
zone hyperplasia and four adolescents with clonal NMZL, they looked for the presence
of H. influenzae. In all patients with hyperplasia, they detected H. influenzae (either
direct culture or highly sensitive PCR), whereas this was lacking in the lymphoma
cases. In only 1/28 control samples, they detected the microorganism, in very low
quantity. Of note, in the hyperplasia cases, they detected areas with light chain
restriction (confirmed by flow cytometry). Since several H. influenzae strains are
known to interact with the constant part of IgD on human B cells, leading to their
polyclonal proliferation and activation, the authors speculate that in vivo stimulation
of IgD+ marginal zone B cells by this bacterium may be implicated in this particular
lymphadenopathy. The results of course indicate to be very careful with the diagnosis
of paediatric NMZL; the methods they used to detect H. influenzae require quite some
expertise and preferably the availability of frozen tissue and is therefore not feasible
for most laboratories.
There are other microorganisms involved in lymphomas like the Epstein-Barr virus (EBV),
hepatitis C and Helicobacter pylori (amongst others). According to Tognon et al. [13],
we need to add simian virus 40 (SV40) to the list. In serum samples from NHL-affected
patients (n = 150) along with controls represented by patients with other cancers
(n = 142) and healthy subjects (n = 300), they found antibodies against SV40 in about
40 % of the NHL patients, 20 % in other cancer patients and 15 % in healthy individuals.
Of course, such an association does not directly proof that SV40 plays a role in the
lymphoma development, but is interesting enough to further study its role.
To have an idea on the outcome of rare lymphomas, there is a need for data from large
registries. Xing et al. [14] analyzed clinicopathologic features for 107 patients
(diagnosed between 1985 and 2012) with splenic MZL (SMZL) which accounts for less
than 2 % of all NHL cases. The median age was 67 years (range 30–88), with 40 % male,
almost all stage IV and with splenomegaly, bone marrow involvement and peripheral
blood involvement (87 %). As initial treatment, 52 underwent splenectomy (10 with
chemotherapy), 38 chemotherapy alone (21 chemoimmunotherapy containing rituximab,
1 rituximab alone) and 2 antivirals for hepatitis C, and 15 were observed only. The
10-year overall survival for first-line splenectomy versus chemotherapy was 61 and
42 %, respectively. The 10-year failure-free survival after first-line splenectomy
versus chemotherapy was 39 and 14 %. Amongst the 38 patients who received first-line
chemotherapy, the addition of rituximab did not result in increased survival. Fifteen
patients transformed to aggressive lymphoma with median time to transformation of
3.5 years (range 6 months to 12 years), and the 10-year transformation rate was 18 %.
The authors conclude that splenectomy remains a reasonable treatment for patients
with SMZL. Parry et al. [15] try to find prognostic markers for this lymphoma type.
In 175 cases, they found recurrent mutations in TP53 (16 %), KLF2 (12 %), NOTCH2 (10 %),
TNFAIP3 (7 %), MLL2 (11 %), MYD88 (7 %) and ARID1A (6 %). KLF2 mutations were early,
clonal events, enriched in patients with del(7q) and IGHV1-2*04 B cell receptor immunoglobulins,
and were associated with a short median time to first treatment. In multivariate analysis,
mutations in NOTCH2 and 100 % germline IGHV gene identity were independent markers
of short time to first treatment, while TP53 mutations were an independent marker
of short overall survival. They conclude that they have demonstrated that NOTCH2 and
TP53 gene mutations are independent markers of reduced treatment-free and overall
survival in SMZL. How this translates to altered clinical decision-making remains
unclear.
Litvinov et al. [16] investigated the geographic clustering of cutaneous T cell lymphoma
(CTCL), which is always quite risky in rare tumours. Using region, zip code, age,
sex and ethnicity, the authors analyzed the demographic data of 1047 patients from
Texas from 2000 through 2012 and another set of 1990 patients who were recorded between
1996 and 2010. There was geographic clustering of patients in three communities in
which CTCL incidence rates were 5 to 20 times higher than the expected population
rate. The authors conclude that their data indicate the existence of yet unknown external
causes/triggers for this rare malignancy. Although the data are relatively large,
still such conclusions have to be drawn very cautiously, since familial clustering,
enhanced awareness and chance may underlie such clustering as well.
Defining entities
B cell lymphomas
A mutation in the MYD88 gene is present in over 90 % of lymphoplasmacytic lymphomas
(LPLs) and since this entity is sometimes difficult to separate from MZ Martinez-Lopez
et al. [17] investigated lymphoma samples from 19 patients with LPL, 88 patients with
SMZL, 8 patients with nodal (N)MZL and 21 patients with extranodal (EN)MZL. Of note,
upon review and integrating mutational, histologic and clinical data, five cases were
reclassified as LPL, reiterating the fact that this diagnosis is not always straightforward.
After reclassification, MYD88 L265P was detected in 13/86 (15 %) SMZL and in 19/24
LPL (79 %) cases. The mutation was absent from NMZL and ENMZL cases. A strong correlation
was found between the presence of an IgM monoclonal paraproteinaemia and the MYD88
L265P mutation. SMZL cases positive for MYD88 L265P were also associated with monoclonal
IgM paraproteinaemia (4/13 cases), although with less serum paraproteinaemia. They
also had a higher frequency of plasmacytic differentiation (9/13) but with no correlation
between the presence of mutation and of light chain-restricted plasma cells in tissue.
The authors conclude that demonstration of the MYD88 L265 mutation is a valuable tool
for the diagnosis of LPL, although some SMZL cases carrying the mutation do not fulfil
the diagnostic criteria for LPL. The MYD88-negative LPL cases, nevertheless, may pose
a challenge to separate from NMZL with plasmacytic differentiation.
Morita et al. [18] addressed the issue of so-called in situ FL. As I discussed in
previous reviews of the literature, this lesion is rare in unselected patient groups
and seems to have little clinical consequences when found as an isolated finding.
The approach of Morita et al. was the other way around: they looked for patients with
FL who had a previous abdominal cancer with lymph node resection and found 4 such
patients from a total of 150 patients with FL. All four had in situ FL in the previously
resected lymph nodes. The time from lymphadenectomy to the diagnosis of FL was 23–120 months.
They conclude that although the rate for development of FL in individuals with in
situ FL is low in prospective studies, their data indicate that follow-up studies
for a longer period are necessary. However, I would certainly not advocate to look
for this lesion with bcl2 staining in lymph nodes from abdominal cancer patients,
since it would result in unnecessary worry of a second malignancy in these cancer
patients while it still seems that the chance that they have clinical relevant disease
is low and there is no place for early treatment.
Some FL cases are CD5 positive, but the meaning of this aberrant expression is not
clear. Li et al. [19] collected 88 such cases (53 men and 35 women; median age, 60 years;
range, 31–86). The lymphoma was diagnosed relatively often at an extranodal (EN) site
(initially in lymph nodes in 66 and EN sites in 22 patients). The presence of t(14;18)(q32;q21)/IGH-BCL2
or another BCL2 translocation was relatively low in 28/44 (64 %) cases. Clinical features
also differed with CD5-negative FL; with a median follow-up of 55 months, 15 patients
died which is rather high; this fits with the relatively high International Prognostic
Index, and commonly development of DLBCL (38/88). They conclude that CD5 expression
in FL is associated with an adverse outcome; however, it remains questionable whether
all these cases really represent FL or may be other lymphoma types with extensive
follicular colonization.
Primary DLBCL of the testis is a lymphoma with specific clinical and pathological
features. Twa et al. [20] performed bacterial artificial chromosome capture sequencing
on three such cases and found novel CIITA, FOXP1 and PDL rearrangements involving
IGHG4, FLJ45248, RFX3, SMARCA2 and SNX29. Immunohistochemistry showed an association
between PDL rearrangements and increased protein expression. Using fluorescence in
situ hybridization (FISH) in a larger series of testicular DLBCL, CIITA (8/82; 10 %)
and FOXP1 (5/74; 7 %) rearrangements appeared to be recurrent. These data suggest
that immune-checkpoint inhibitor therapy might be a promising intervention for this
rare lymphoma type that develops in an immune-privileged site.
T cell lymphomas
ALK-positive and ALK-negative ALCL share morphological features but differ in prognosis
and, obviously, ALK expression as a result from the t(2;5) translocation. Steinhilber
et al. [21] compared the microRNA (miRNA) expression between these two types. Their
NGS approach identified 106 significantly differentially expressed miRNAs. The top
significantly differentially expressed miRNAs included five upregulated miRNAs: miR-340,
miR-203, miR-135b, miR-182 and miR-183 and seven downregulated miRNAs: miR-196b, miR-155,
miR-146a, miR-424, miR-503, miR-424* and miR-542-3p. The miR-17-92 cluster was also
upregulated in ALK+ cells. Additionally, they identified a signature of three miRNAs
significantly regulated by the transcription factor C/EBPβ, which is specifically
overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which
regulates T cell differentiation and modulates T cell receptor (TCR) signalling strength,
was significantly downregulated in ALK+ ALCL cases. These data reveal a miRNA signature
linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR
antigen expression known in ALK+ ALCL. Merkel et al. [22] take this work further.
They also found that ALK-positive ALCL, in contrast to ALK-negative ALCL, displays
low miR-155 expression, and discovered that this is due to miR-155 promoter methylation,
but there was no direct effect of the ALK kinase on miR-155 levels. Induced overexpression
of miR-155 in ALK-positive ALCL cell lines resulted in reduced levels of C/EBPβ and
SOCS1. In murine engraftment models of ALK− ALCL, anti-miR-155 mimics are able to
reduce tumour growth and increase levels of cleaved caspase-3 and SOCS1 resulting
in suppression of STAT3 signalling. These data suggest that miR-155 can act as a tumour
driver in ALK-negative ALCL, and blocking miR-155 could be therapeutically relevant.
The data also indicate that ALK-positive ALCL and ALK-negative ALCL are biologically
really different entities.
Cutaneous lymphomas
Cutaneous follicle centre cell lymphoma (CFCL) is regarded as a completely different
disease than FL, although FL may be manifested in the skin. Several studies have shown
that CFCL lacks a bcl2 rearrangement in contrast to FL. Pham-Ledard et al. [23] confirm
this knowledge by analyzing 47 CFCL, 4 of which carry a bcl2 break by FISH, and 6
FL presenting with skin involvement, 3 of which have a bcl2 break. Since some of the
CFCL with a bcl2 break had systemic disease and poor prognosis, one may doubt the
diagnosis in these cases.
Pitfalls in lymphoma diagnosis
The role of EBV in lymphoproliferation in patients with various forms of immune suppression
is well known, but it remains a challenge when to investigate for EBV and what to
do with a positive result. In patients with inflammatory bowel disease (IBD), several
case reports indicate an increased risk in EBV-positive lymphoproliferations, but
it is unclear how aggressive this disease is. Nissen et al. [24] collected biopsies
from 58 patients with IBD who had been tested for the presence of EBV, 28 of which
were positive. The presence of EBV was correlated with an atypical infiltrate, which
was present in more than 50 % of the EBV-positive cases and only one of the EBV-negative
cases. Two EBV-positive patients had overt lymphoma, and eight had a monomorphic infiltrate.
After reducing immune suppression, the morphology returned to normal, although one
of the two lymphoma patients had in addition chemotherapy. No patient died from the
lymphoproliferation, but four EBV-positive patients underwent colectomy due to severe
complaints. These data indicate that the presence of EBV-positive lymphoproliferation
in IBD can be suspected based on morphology, but that it is less aggressive compared
to other immune-suppressed situations.
Prognostic factors in lymphoma
Also this time, there is a small selection of articles that deal with prognostication
in NHL. I chose those articles that have high-quality data, including a discovery
and a confirmation cohort and well-defined relevant patient selection.
In FL, Kridel et al. [25] applied IHC for CD68 and CD163 to two large tissue microarrays
(TMAs): one with samples from 186 patients from the BC Cancer Agency (BCCA) and the
other with 395 samples from the PRIMA trial patients. Both groups had been treated
with rituximab, but within different chemotherapy regimes. Macrophage infiltration
was assessed using image analysis, and an increased CD163-positive pixel count was
predictive of an adverse outcome in the BCCA dataset (5-year progression-free survival
38 versus 72 % in the training cohort and 29 versus 61 % in the validation cohort).
However, in the PRIMA trial, an increased CD163 pixel count was associated with favourable
outcome (60 versus 44 % in the training cohort and 55 versus 37 % in the validation
cohort). These data clearly indicate the importance to correlate a prognostic factor
to well-defined patient groups, but also that it is difficult to introduce such a
marker in clinical practice.
miRNA profiling is very well feasible on paraffin-embedded tissue specimens, and therefore,
many studies are done using this technique. A good example is the work by Husby et
al. [26] who performed genome-wide miRNA microarray profiling of 74 diagnostic MCL
samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated
in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation
cohort). Three miRNAs (miR-18b, miR-92a and miR-378d) were significantly differentially
expressed in patients who died of MCL in both cohorts. miR-18b was superior to miR-92a
and miR-378d in predicting high risk. Transfection of two MCL cell lines with miR-18b
decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b
may render MCL cells resistant to chemotherapy by decelerating cell proliferation.
Based on these results, they generated a new biological MCL International Prognostic
Index (MIPI-B)-miR prognosticator, which improved the identification of high-risk
patients with regard to cause-specific, overall and progression-free survival.
The lymphoma on which most prognostic work is done is DLBCL. Shepshelovich et al.
[27] performed miRNA profiling but had only 83 samples, predefined in favourable and
poor prognosis, with a validation set of only 13 cases. The approach resulted in significant
differences, but the findings did not add to the standard prognostic indicators. Knudsen
et al. [28] took a more thorough approach. They developed a miRNA response predictor
in DLBCL based on the measured growth inhibition of 60 human cancer cell lines (NCI60)
in the presence of doxorubicin, cyclophosphamide, vincristine and etoposide as well
as the baseline microRNA expression of the 60 cell lines. This response predictor
consisting of 20 microRNAs was blindly validated in a cohort of 116 de novo DLBCL
patients treated with R-CHOP or R-CHOEP as first-line treatment. The predicted sensitivity
based on diagnostic FFPE samples matched the clinical response. When the International
Prognostic Index (IPI) was included in the prediction analysis, the separation between
responders and non-responders improved. The predictions were almost the same when
diagnostic biopsies were used as when relapse biopsies were used. These preliminary
findings warrant testing in a larger cohort of relapse patients to confirm whether
the miRNA-based predictor can select the optimal second-line treatment and increase
survival.
MYC status is a relevant issue in DLBCL, but the impact varies amongst the subtype
of lymphoma and the type of MYC alteration. Caponetti et al. [29] investigated the
significance of MYC, BCL2 and BCL6 gene abnormalities in a cohort of 205 DLBCL patients
studied by conventional and/or fluorescence in situ hybridization cytogenetic analysis.
One hundred seventy-two cases (84 %) were classified as MYC−, 17 (8 %) were MYC+/BCL2−/BCL6−,
and 16 (8 %) were double/triple-hit lymphomas (i.e. MYC+/BCL2+, MYC+/BCL6+ or MYC+/BCL2+/BCL6+).
They found a significant difference in event-free survival amongst the three groups,
with the double/triple-hit group having the worst outcome. Somewhat surprising, patients
who were MYC+, but BCL2− and BCL6−, do not have a worse outcome when compared to those
who are MYC− which is in contrast with existing data in the literature. Li et al.
[30] looked for MYC protein expression in primary mediastinal DLBCL, although these
cases rarely have a genetic event that includes a MYC alteration. They found variable
MYC protein expression by IHC in 30 (94 %) of 32 cases including 10 cases with high
MYC IHC expression of at least 30 % positive nuclei. FISH analyses for MYC rearrangement
on these 10 cases were negative. Review of clinical data of a subset (!) of cases
with high- and low-MYC IHC expression showed no differences in clinical outcome. These
results are somewhat difficult to interpret, but actually, the numbers are too low
to draw meaningful conclusions. Even the work of Wang et al. [31], which has findings
that are contradictory to these results, lacks sufficient power, although they study
135 cases of DLBCL. They analyzed the differences between genetic double-hit lymphomas
and those defined on protein expression. They determined in their cohort in whom MYC/8q24
and BCL2/t(14;18)(q32;q21) statuses were assessed by FISH at diagnosis of MYC and
BCL2 expression by immunohistochemistry. A total of 54 (40 %) cases were positive
for both MYC and BCL2, supporting a diagnosis of double-hit lymphoma, which is a quite
high number compared to those of the literature. Amongst them, 19 (35 %) had MYC rearrangement,
12 (22 %) had multiple copies of MYC, 19 had no MYC abnormalities, and in 4 cases,
FISH analysis failed. BCL2 abnormalities were present in 28/54 (52 %) cases (20 rearranged
and 8 multiple copies). MYC rearrangement correlated with a significantly worse overall
survival, whereas BCL2 genetic status did not correlate with survival. They conclude
that MYC and BCL2 expression by immunohistochemistry correlates with gene status by
FISH, but that immunohistochemistry is neither specific nor adequately sensitive to
be used as a surrogate for MYC and BCL2 gene status using any cut-off level.
Enteropathy-associated T cell lymphoma (EATL) has a particularly poor prognosis, and
the International Prognostic Index and the prognostic index for peripheral T cell
lymphoma have limited predictive value for outcome. De Baaij et al. [32] performed
a retrospective multicenter study, based on 92 patients. They were able to distinguish
three risk groups: a high-risk group, characterized by the presence of B symptoms
(median overall survival of 2 months); an intermediate-risk group, comprising patients
without B symptoms and an IPI score ≥2 (7 months); and a low-risk group, representing
patients without B symptoms and an IPI score of 0–1 (34 months). They conclude that
their new validated prognostic model (EPI) accurately predicts survival outcome in
EATL and may be used for patient selection for new therapeutic strategies and evaluation
of clinical trials.
Ancillary techniques
Technological development is one of the drivers of modern pathology. New methods therefore
need to be evaluated all the time to see whether they can improve standard pathology.
Liang et al. [33] retrospectively analyzed EBV status in 232 DLBCL patients using
EBV-encoded small RNA (EBER) in situ hybridization (ISH) and EBV DNA analysis in whole
blood. EBER was positive in 24 (10 %) patients, and EBV DNA was positive in 18 (10 %)
patients; the two analyses had more than 90 % concordance. EBV-positive patients as
determined by both techniques had worse overall survival than those without EBV positivity.
The additional value of blood testing above tissue testing lies in the following:
the transformation from positive to negative after cycle 3 with chemotherapy had the
most capacity to distinguish a superior from an inferior outcome. These findings suggest
that EBV DNA in whole blood has good concordance with EBER ISH and that it may be
a good biomarker for monitoring the disease.
Although clonality testing is already a high level, high-quality test, the development
of next-generation sequencing may have its impact in this area of pathology, too.
Appenzeller et al. [34] developed a next-generation sequencing approach on the ion
torrent personal genome machine to characterize the immunoglobulin heavy gene V-D-J
rearrangements. The method was applied to two diagnostic tissue samples, including
formalin-fixed and paraffin-embedded tissue, of two patients with iatrogenic immunodeficiency-associated
Epstein-Barr virus lymphoproliferative disorder, with ulcerative colitis as underlying
disease. The immunoglobulin rearrangement sequences obtained by next-generation sequencing
revealed undoubtedly clonally related lesions in two tissue biopsies that were taken
over time in the first patient, which is concordant with disseminated lymphoma. The
other patient showed two clonally unrelated lesions, which is incompatible with clonal
dissemination. This information was not inferred from evaluation of the heavy and
light chain rearrangements by fragment analysis, which is currently the gold standard.
This study nicely demonstrates the diagnostic application of next-generation sequencing
of immunoglobulin rearrangement assessment in pathology for clinical decision-making
in patients with several simultaneous or subsequent lymphoproliferations. Another
study using a next-generation sequencing approach investigated a different potential
use. Kurtz et al. [35] prospectively evaluated its utility in 311 blood and 105 tumour
samples from 75 patients with DLBCL, comparing the cellular (circulating leukocytes)
and acellular (plasma cell-free DNA) compartments of peripheral blood. The results
were related to clinical outcomes and 18FDG PET/CT (n = 173). Clonal immunoglobulin
rearrangements were detected in 83 % of patients with adequate tumour samples to enable
subsequent monitoring in peripheral blood (which is lower than when the complete Biomed
set is used). Molecular disease measured from plasma, as compared to circulating leukocytes,
was more abundant and more correlated with radiographic disease burden. Prior to treatment,
molecular disease was detected in the plasma of 82 % of patients compared to 71 %
in circulating cells. However, molecular disease was detected significantly more frequently
in the plasma at time of relapse (100 vs. 30 %). Detection of molecular disease in
the plasma often preceded PET/CT detection of relapse in patients initially achieving
remission. This method, therefore, is promising, as confirmed by Roschewski et al.
[36] who performed similar work. Clonal products were identified in pretreatment specimens
from 126 patients who were followed up for a median of 11 years. Interim monitoring
of circulating tumour DNA at the end of two treatment cycles in 108 patients showed
a 5-year time to progression of about 40 % in patients with detectable circulating
tumour DNA and 80 % in those without detectable circulating tumour DNA. So, also this
study indicates that surveillance of circulating tumour DNA identifies patients at
risk of recurrence before clinical evidence of disease.
Already more than 10-year DLBCL is subdivided into germinal centre B cell-like and
activated B cell-like subtypes. Still, these lymphomas are difficult to differentiate
in routine diagnosis, impeding the use of this subtyping in clinical decision-making.
Mareschal et al. [37] therefore developed a simple and rapid classifier based on a
reverse transcriptase multiplex ligation-dependent probe amplification assay and 14
gene signatures. Compared with the Affymetrix U133 + 2 gold standard, all 46 samples
of a validation cohort classified using both techniques were attributed to the expected
subtype. Similarly, 93 % of the 55 samples of a second independent series characterized
with a mid-throughput gene expression profiling method were classified correctly.
The developed assay was sensitive enough to obtain reliable results from formalin-fixed,
paraffin-embedded samples and flexible enough to include prognostic factors such as
MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall and progression-free
survival differences between the two subtypes were confirmed. As the authors conclude,
because the multiplex ligation-dependent probe amplification method is already in
use and requires only common instruments and reagents, it could easily be applied
to clinical trial patient stratification to help in treatment decisions.