Evaluation of the Prognostic Importance of c-Myc and Bcl-2 Expressions and the Presence of Epstein-Barr Virus in Classical Hodgkin Lymphoma
Aydan KILIÇARSLAN1 , Vedia ÖZTÜRK2, Aysun ÞENTÜRK YIKILMAZ3, Mehmet DOÐAN4, Mesut AKYOL5
1Department of Pathology, Ankara Yýldýrým Beyazýt University, School of Medicine, ANKARA, TURKEY
2Van Education and Research Hospital, VAN, TURKEY
3Department of Hematology, Ankara Atatürk Education and Research Hospital, ANKARA, TURKEY
4Department of Pathology, Ankara Dr. Abdurrahman Yurtaslan Oncology Education and Research Hospital, ANKARA, TURKEY
5Department of Biostatistic, Ankara Yýldýrým Beyazýt University, School of Medicine, ANKARA, TURKEY
Keywords: Hodgkin lymphoma, c-Myc, Bcl-2, EBER
Although classical Hodgkin lymphoma (cHL) has a relatively good prognosis, it also entails different treatment responses and involves
patients who have different clinical courses. Our aim was to investigate c-Myc, Bcl-2 and EBV biomarkers in cHL and their relationship with the
Material and Method: c-Myc and Bcl-2 immunohistochemical staining with EBER in situ hybridization (EBER-ISH) was applied to the
paraffin sections of 94 cases diagnosed as cHL. These cases were classified into two groups as low and high clinical symptoms according to the
International Prognostic Scores (IPS).
Results: Positive results were obtained in 83 (88.3%) cases with c-Myc and 39 (43.5%) cases with Bcl-2 while EBER-ISH was found positive in 42
(44.7%) cases. No difference was found between the groups of low/high IP scores with respect to the positive or negative results of EBER-ISH,
Bcl-2 and c-Myc. When Bcl-2 and c-Myc positive cases were grouped together and compared to the IP scores of the remaining cHL cases, again
no difference was seen. Extranodal involvement and bone marrow involvement was observed in 25 (26.5%) and 9 (9.5%) cases, respectively.
Similarly, no statistically significant differences was found between these groups according to their positivity with EBER-ISH, Bcl-2 and c-Myc.
Conclusion: We could not find any relationship between Bcl-2, c-Myc and EBER-ISH positivity and the low/high IPS groups in cHL. New studies
with larger series are needed in which more precise cut-off values are used and clinically and biologically heterogeneous groups of cHL patients
are determined more clearly.
Hodgkin lymphoma (HL) is a partially homogeneous
neoplastic disorder due to its relatively indolent clinical
course (1). However its histomorphological findings,
immune phenotype, genotype, relationship with viruses
and its responses to treatment may vary (2-7). Several
clinical parameters (age, stage, subtype, tumor burden,
etc.) are used as predictive factors in the HL patients’
lifespan and responses to treatment (8-10). Until now,
genetic and molecular steps in the progression of HL
have not been completely understood. Genetic and
immunohistochemical studies have been conducted into
multiple cell cycle regulators in the pathogenesis of HL.
These studies include numerous biological markers such
as apoptosis inhibitors (Bcl-2, bcl-X), tumor suppressors (p-53, RB, etc.), proto-oncogenes (c-Myc, NOTCH1, IRF4,
etc.), as well as the Epstein-Barr virus (11-18).
Certain biological markers are known to be predictive
factors for success of treatment and recovery chance
of some non-Hodgkin lymphoma (NHL) types. It has
been shown that the presence of Bcl-2, c-Myc, and Bcl-6
expressions in diffuse large B-cell lymphomas are related
with poor prognosis (19,20) and the presence of p-53
mutation in mantle cell lymphomas is a predictor of
aggressive behaviour (21,22). It has also been determined
that presence of EBV in T-cell NHLs is related with poor
Several prognostic models are used to classify lymphoma
patients into groups of high and low risk. The International
Prognostic Score (IPS) consists of a combination of various clinical and laboratory parameters and the risk of a patient
is determined by these parameters (25,26). In fact, even in
risk groups as determined by IPS, different treatment results
may be obtained. Some researchers have tried to develop
research models that use biological markers together with
clinical prognostic models. Nevertheless, the independent
prediction accuracy of these indicators is limited and not
In this study, we aimed to put forward c-Myc, Bcl-2 and
EBV status of cHL and whether these indicators are related
with prognostic findings as determined by IPS.
Ninety-four cases diagnosed as classical Hodgkin
Lymphoma (cHL) between 2007 and 2016 were included in
the study. The cases were re-evaluated by two pathologists
(A.K. and V.Ö.) according to the 2017 WHO classification
criteria. Diagnostic blocks with cHL were chosen for
immunohistochemical staining and EBER (Epstein-Barr
virus-encoded RNA) in situ hybridization tests. Clinical
parameters of prognostic importance were obtained
through the computer files and data in the hematology and
oncology clinics where these patients were monitored.
Clinicopathological data (age, sex, histological subtype,
clinical stage, bone marrow involvement, prognostic score,
the presence and date of relapse, together with the presence
of extranodal involvement) of the cases were obtained
from the patient files and were categorised by using IPS
(International Prognostic Score). IPS scores were coded as
0, 1, and 2 representing “low” and 3 and above representing
3-4 μm thick sections were placed on slides covered
with poly-L-lysine out of the blocks of cHL-diagnosed
formalin-fixed paraffin-embedded biopsy samples for
immunohistochemical studies. The standard protocol
was applied by the Benchmark GX IHK/ISH (Ventana)
automatic staining device for the anticors of c-Myc (clone
Y69, Ventana) and Bcl-2 (clone 2/100/05, Novocastra). For
both of the markers, stainings below 40% were regarded as
“negative” and those above 40% were regarded as “positive”
in Hodgkin/Reed-Sternberg (HRS) cells.
EBER (EBV-encoded RNA) in Situ Hybridization
EBER-ISH was applied to all cases using ISH iVIEW Blue
Detection Kit (Ventana) with silver labeled oligonucleotide
probes (INFORM EBER Probe, Ventana) in the Benchmark
GX IHK/ISH (Ventana) autostainer device. After
deparaffinization and soft and standard cell healing, 12 minutes of protease 3, and the INFORM EBER steps were
applied; then 6 minutes of red counterstain was performed
on the background and dark-blue nuclear staining was
considered to be positive in HRS cells.
The SPSS for MacOS program was used to analyse the data.
Discrete numeric variables of descriptive statistics were
shown as mean±standard deviation or median (minimummaximum),
nominal variables were shown as the number
of cases and percentage (%).
Nominal variables were evaluated by using Pearson’s Kisquare
or Fisher’s exact test. The results for p<0.05 were
regarded as statistically significant.
Samples taken from a total of 94 patients of whom 33
were female (35.1%) and 61 were male (64.9%) and whose
median age was 40.5 years (ranging between 15 and 75
years). Of the cases, 55 (58.5%) were nodular sclerosing, 19
(20.2%) were mixed cellular, 15 (16.0%) were lymphocyte
rich and 5 (5.3%) of them were lymphocyte poor subtype.
Examined IPS scores were ranged between 0 and 7, and
the median IPS score was 2.0 (IQR=2.0). 63 (67.0%) of the
cHL cases were classified as low and 31 (32.9%) as high risk
While the staining rate for c-Myc ranged between 0%
and 100%, the staining median for c-Myc was calculated
as 70.0% (IQR=20.0) (Figure 1A-B). The Bcl-2 staining
ranged between 10% and 100%; and staining median was
0% (IQR=60%) (Figure 2).
Click Here to Zoom
|Figure 1: A) In Bcl-2 immunohistochemical staining, lymphocytes around HRS cells shows positive staining in the form of rosettes (IHC;
x40). B) Cytoplasmic positive staining in HRS cells with Bcl-2 (IHC; x100).
Click Here to Zoom
|Figure 2: In c-Myc immunohistochemical staining, common
positive staining is observed in HRS cells’ nucleus (IHC; x400).
Values below 40% were regarded as “negative” while
40% and above were regarded as positive for both of the
markers. Accordingly, 83 (88.3%) cases with c-Myc were
seen to be positive while 39 (41.5%) cases with Bcl-2 were
evaluated as positive. EBV was determined as positive in 42
(44.7) cases (Figure 3).
Click Here to Zoom
|Figure 3: In EBER ISH, dark-blue nuclear staining was considered
to be positive in HRS cells (CISH; x400).
No statistical significant difference was found, concerning
EBV, Bcl-2 and c-Myc positivity between the low and high
risk groups of IPS (p>0.05) (Table I).
Click Here to Zoom
|Table I: The staining rates with c-Myc, Bcl-2 and EBER-ISH in IP score groups
Extranodal involvement was found in 25 (26.5%) of the
cases. No difference was found about Bcl-2 and c-Myc
staining rates with relation to extranodal involvement
(p>0.05) (Table II). However, 21 (84%) of 25 cases with
extranodal involvement also had positive staining with
c-Myc. The rate of extranodal involvement in EBV positive
cases was significantly higher than in EBV negative cases
(χ 2 =5.413; p=0.023). When all other factors (such as age, gender and the stage of illness) are assumed stable, the
risk of extranodal involvement in EBV positive cases is
OR=2.98 ( 95% CI: 1.16 - 7.65) times higher than in EBV
Click Here to Zoom
|Table II: The staining rates of c-Myc, Bcl-2 and EBER-ISH in relation to extranodal involvement
Bone marrow involvement was seen in 9 (9.5%) of the
cases. No difference was found concerning the staining
rates of c-Myc, Bcl-2 and EBER-ISH in relation to bone
marrow involvement (p>0.05) (Table III). Positive stainig with c-Myc was found in 7 of the 9 cases with bone marrow
involvement, and positive Bcl-2 was found in 3 of them.
Click Here to Zoom
|Table III: The staining rates of c-Myc, Bcl-2 and EBER-ISH in relation to bone marrow involvement
EBV was found to be positive in 42 (44.6%), and negative
52 (55.3%) of the cases with EBER-ISH. No significant
relationship was detected between EBER-ISH, c-Myc and
Bcl-2 expressions (Table IV).
Click Here to Zoom
|Table IV: The staining rates of c-Myc and Bcl-2 in relation to the EBER-ISH positivity
To observe any difference according the IP scores between
Bcl-2+/c-Myc+ patients and the remaining patients, cases were reclassified into two groups: Bcl-2+/c-Myc+ cases and
other cases (Bcl-2+/c-Myc-; Bcl-2-/c-Myc-; and Bcl-2-/c-
Myc+) (Table V). There was no difference between the low
and high IPS groups (p>0.05).
Click Here to Zoom
|Table V: The staining rates for IPS groups of Bcl-2 +/c-Myc + and the others
A considerable number of HL cases are tumors that respond
to first-line therapy (27). However,, in approximately 20%
to 30%, relapse, treatment complications and death from the
disease occur (28, 29). Additionally, a sufficient response to
standard treatment could not be obtained in more than one
third of HL cases. The factors affecting treatment response
in this patient group may include advanced stage disease,
the presence of B symptoms, oncogenic proteins, or
molecular abnormalities in the suppressor protein panels.
In several studies, a comparison has been made between
the biological markers that have potential routine use and
the clinical results and the effects on known prognostic
factors have been researched (3,30,31).
Several proto-oncogenes may affect lymphoid malignancies
and one of the most important of these is c-Myc (32). c-Myc
plays role in various cellular functions such as cell cycle, cell
growth, cell metabolism, biosynthesis, adhesion and also
the control of mitochondrial function. The determination
of c-Myc protein expression and gene translocation is
important in the diagnosis of lymphomas and specifying
clinical sequence of aggressive B cell lymphomas (33,34). It
has been demonstrated that c-Myc translocation is related
with a poor prognosis in the subgroups of diffuse large B
cell lymphoma patients (20,35). Additionally, it has also
been shown that diffuse large B-cell lymphoma patients
with co-expression of c-Myc, Bcl-6 and Bcl-2 proteins
have a poor prognosis and do not respond to routine
chemotherapy (19,20,35). The presence of c-Myc has been
searched in a limited number of studies concerning HL
and positive staining was shown for the major part of those
cases (36,37). We aimed to show c-Myc expression status
in cHL and to search whether there is any risk in terms of
IPS risk groups. However, we were unable to obtain any
significant difference between the low and high risk IPS
groups. Besides, in 61 (68.5%) of 89 cases, IPS was found to
be low. Moreover, there was no bone marrow involvement in 82 (92.1%) and no extranodal involvement in 68 (76.4%)
out of these 89 cases.
Biological markers such as Bcl-2, BAX, Bcl-X and p53
are proteins that organize apoptosis. High staining rates
of Bcl-2 in HRS cells have been associated with negative
consequences in HL patients in some studies (5, 12, 38).
In our study, we did not find any significant relationship
between Bcl-2 expression in HRS and the low and high
risk IPS groups. Furthermore, 25 (64,1%) out of 39 Bcl-2
positive cases were grouped as low risk IPS. Also, out of
39 cases that were stained Bcl-2 positive, 27 (69.2%) and
36 (92.3%) cases had no extranodal involvement and
no bone marrow involvement, respectively. The chosen
method and the cut-off value regarding positivity may
have resulted differently in previous studies. There aren’t
many articles concerning the staining threshold of c-Myc
and Bcl-2 expressions in HL. In certain studies mentioning
significant differences, 10% has been used as a threshold
for Bcl-2 positivity, regarding the clinical consequences of
HL (3,5,38). In studies that assumed the threshold as 20%
and above, no relationship has been found in the same
context (12,39). However, diffuse large B-cell lymphoma
studies concerning c-Myc and Bcl-2 expression, usually
40% and above positivity is used as cut-off point (40,41).
Hence, we preferred to use the same scope for our study,
but this may be the cause that why we could not find any
significant relationship between Bcl-2/c-Myc expressions
and IP scores. This leads to stating the limitation of our
study, not including either applied therapy or the results of
the treatment for the patients .
In the literature, the Epstein-Barr virus (EBV) has been
determined to be positive in 47.9% of cHL patients (42).
Whether there is a relationship between EBV infection
and the advanced clinical stages of cHL is highly debatable.
While some studies (43-45) have found an important
correlation between EBV and the advanced stage of cHL,
other studies have not found such a relationship (46-48).
We found that 42 (44.7%) of cHL cases were EBV positive.
We could not find any difference between positive and
negative EBV cases in terms of IPS and bone marrow
involvement, but, there was significantly high extranodal involvement in those cases which were EBV positive.
Besides, no relationship was found between EBER-ISH and
Bcl-2/c-Myc expressions in our series. These markers have
been defined as independent factors from each other in
other literature (6,15,49).
Finally, we also researched whether co-expression of c-Myc
and Bcl-2 is related with low and high risk IP scores, but we
could not find any significant result.
It is concluded that future studies with larger series may
lead to finding an optimal cut-off value for c-Myc and Bcl-
2 and can provide more prognostic information in cHL,
including determining treatment-resistant/high-risk cases.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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