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DOI:
10.4103/2395-4469.177565
Livin expression by semi‑quantitative
immunofluorescent staining in Hodgkin
lymphoma: A promising marker or a
leading role in pathogenesis?
Amin Ziaei1,2, Amir Pouya Tanhaei1,2, Ahmad Reza Salehi Chaleshtori3,
Safoura Mazrouei4, Mona Kharaji2, Kianoosh Keyhanian5, Mansoor Salehi1,3
Abstract:
Introduction: A novel human inhibitor of apoptosis protein family member, termed livin, is enrolled in pathogenesis
of different human malignancies and it was studied as a potential treatment target for malignancies. Methods: This
study was designed to explore the clinical significance of livin expression in Hodgkin’s lymphoma (HL). We
evaluated livin expression in 78 paraffin‑embedded blocks including 39 stage defined cases with defined stages
of HL and 39 control subjects. Tissue microarray‑based semi‑quantitative immunofluorescent staining was
applied for protein expression profiling in both infiltrating nonneoplastic cells and neoplastic cells of cases and
control samples. Results: Our results represented that the mean ratio (M.R) of livin/glyceraldehyde 3‑phosphate
dehydrogenase (GAPDH) expression was significantly increased between infiltrating background cells in HL and
control cases (0.54596 vs. 0.50827, P < 0.001). In addition, significant difference was found in M.R of livin/GAPDH
expression in neoplastic cells regarding the background cells in tumor microenvironment (0.59024 vs. 0.54596,
P < 0.001). In addition, this study confirmed a significant increase of livin expression through more advanced stages
of HL (0.52888 vs. 0.580146, P < 0.01). Conclusion: These findings recommended a possibly critical role of livin
in pathogenesis of HL. It also could be a novel prognostic marker and a potential therapeutic target in this disease.
Key words:
Department of
Genetics and Molecular
Biology, School of
Medicine, Isfahan
University of Medical
Sciences, 2Medical
Students Research
Center, School of
Medicine, Isfahan
University of Medical
Sciences, 3Medical
Genetic Center of
Genome, Isfahan,
Iran, 4Faculty of
Medicine, Friedrich
Schiller University of
Jena, Jena, Germany,
5
Ottawa Hospital
Research Institute,
Ontario, Canada
1
Address for
correspondence:
Dr. Mansoor Salehi,
Isfahan University of
Medical Sciences, Hezar
Jirib Street, Isfahan, Iran.
E‑mail: m_salehi@med.
mui.ac.ir
Submission: 15‑08‑2015
Accepted: 05‑12‑2015
Apoptosis, Hodgkin lymphoma, immunofluorescent staining, livin protein
Introduction
H
odgkin lymphoma (HL) is the most striking
example of tightly tumor‑host correlation,[1]
and its classic form is responsible for 95% of
the cases.[2] Classic HL is sub‑divided into four
entities such as nodular sclerosis classical HL,
mixed cellularity classical HL, lymphocyte‑rich
classical HL, and lymphocyte depleted classical
HL. [3] The remaining 5% represent nodular
lymphocyte predominant HL (NLPHL).[2,3]
Unlike most cancers, malignant cells in HL
are outnumbered by background cells in the
microenvironment of tumor.[2,4] This accounts for
only around 1% of all cells in the tumor tissue.[1,2,4]
In addition, as for HL, major reactive cells in
lymph nodes are lymphocytes (mostly T‑cell),[5]
macrophages/histiocyte cells, eosinophils, mast
cells, plasma cells, fibroblast, and neutrophils.[5,6]
Despite increasing attention to HL, its molecular
biology is still poorly understood [2,7] and
supplementary studies are required to discover
unresolved molecular pathobiological issues
of HL (details described elsewhere).[2,8] Some
advances in molecular profiling of Hodgkin and
Reed‑Sternberg (HRS) cells revealed the potential
© 2016 Oncobiology and Targets | Published by Wolters Kluwer - Medknow
role of these cells in pathogenesis of HL. This
capability is manifested via secretion of cytokines
and chemokines[2,4,9] and eventually escape from
apoptosis.[9,10]
Apoptosis is essentially important in
n e u r o d e g e n e r a t i o n , [8‑10] c a n c e r , a n d
hematolymphoid neoplasm. Progression
of hematological malignancies is related to
impaired regulation of apoptosis. [10‑14] Bcl‑2
and inhibitor of apoptosis proteins (IAPs)
are recognized as hallmarks of apoptosis
regulation [13] and key players in mentioned
malignancies. Upregulation of IAPs correlates
with poor prognosis in a variety of cancers such
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For reprints contact: [email protected]
How to cite this article: Ziaei A, Tanhaei AP,
C h a l e s h t o r i A R , M a z r o u e i S , Kh a r a j i M ,
Keyhanian K, et al. Livin expression by semiquantitative immunofluorescent staining in Hodgkin
lymphoma: A promising marker or a leading role in
pathogenesis?. Oncobiol Targets 2016;3:1.
1
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Ziaei, et al.: Livin expression in Hodgkin lymphoma
as hematological malignancies.[14] The neuronal apoptosis
inhibitory protein (NAIP)/BIRC1, cIAP‑1/BIRC2, cIAP‑2/
BIRC3, X‑linked inhibitor of apoptosis/BIRC4, SURVIVIN/
BIRC5, BRUCE/BIRC6, ILP‑2/BIRC8, and livin/ML‑IAP/
BIRC7 are eight identified IAP members in human.[11,15] Livin
plays a prominent role in apoptosis inhibition.[16] It consists
of a single baculoviral IAP repeat (BIR) domain and RING
finger motif which forms a globular conformation (conserved
by four α‑helices) and three‑stranded anti‑parallel β‑sheets,
respectively.[17,18] It was reported that livin directly interacts
with executioner caspase‑3 and caspase‑7 in vitro and initiator
caspase‑9 in vivo through its BIR domain.[18,19] BIR domain has
a critical role in IAP function and mutations in this domain
decline inhibition of caspase‑9 and general anti‑apoptotic
capability of cell.[20] In addition, truncated form of livin (tLivin)
not only loses its anti‑apoptotic effect but also acquires a
pro‑apoptotic effect.[17,21]
There are some evidence for elevated levels of livin
expression in colon cancer,[22] bladder cancer,[23] lung cancer,[24]
neuroblastoma,[25] pancreatic cancers,[26] osteosarcoma, renal
cell carcinoma,[17] and leukemia including colon cancer[27] and
certainly non‑HL.[10,28]
There is no report about the quantity of livin expression in
HL and discussed the prognostic significance of livin as an
informative biomarker in HL. In addition, there are some
controversial reports around prognostic importance of livin
expression in different malignancies. Hence, the present
study was designed to examine the livin expression in HL
and evaluate the potential role of this molecule as a promising
marker in different stages of HL.
Materials and Methods
Patients and tissue blocks
Livin expression was examined in 78 paraffin‑embedded blocks
including 39 HL Formalin‑Fixed Paraffin‑Embedded (FFPE)
tissue blocks and 30 and 9 FFPE with diagnosed normal and
reactive hyperplasia lymph nodes, respectively. The lymph
nodes were excised in the department of surgery, Seyed Al
Shohada and Alzahra Hospitals, Isfahan University of Medical
Sciences, 2008–2011.
Based on histological examination, primary diagnosis of CHL
was performed through H and E staining and morphologically
confirmed RS cells. Based on immunohistochemical assessment,
RS cells (to consider CHL cases) were verified. Subsequently,
suitable lymph node blocks were confirmed morphologically
with supplementary immunohistochemical staining (data
were not shown).
Clinical features of all patients including sex, age,
B‑symptom (fever, night sweats, and weight loss), lymph
node involvement sites (cervical, axillary, mediastinum,
para‑aortic, perihilar, and inguinal), and extra‑lymphatic
organ involvement (lung, liver, spleen, and bone marrow)
were documented. The clinical staging was performed based
on Ann Arbor staging procedure criteria. In addition, we
determined another staging style which assumed stage I and II
as early‑stages and stage III and IV as advanced‑stages.[29] Some
cases did not have the required clinical information for staging,
2
so they were considered in undefined stage group. Informed
consent was obtained from all donor materials.
Immunostaining
FFPE samples were subjected to histological studies using
standard method. Histological sections (3 μm) were cut
using a Jung rotary microtome (Wetzlar, Germany), floated
out on a 50–50 volume mixture of absolute ethanol‑distilled
water in 48°C, and mounted on glass microscopic slides
which were previously coated with (3‑Aminopropyl)
triethoxysilane (Sigma‑Aldrich, USA) prior to overnight
storage at 37°C.
The semi‑quantitative immunofluorescent staining method used
in this study was described elsewhere.[30,31] Expression of livin
and glyceraldehyde 3‑phosphate dehydrogenase (GAPDH)
proteins was measured by semi‑quantitative immunofluorescent
assay. We used livin primary antibody of BIR detecting amino
acids 264–280 of the short form and 281–298 of the long form of
human livin. This sequence is identical between α and β isoforms
of the livin protein. Therefore, the antibody detects both α and β
isoforms of intact livin protein but not truncated livin proteins.
GAPDH primary antibody was GAPDH mouse anti‑Human.
Fluorochrome‑conjugated secondary antibody against the
species of the anti‑livin and anti‑GAPDH antibodies were
fluorescein isothiocyanate (FITC) affini pure goat anti‑rabbit,
immunoglobulin G (IgG) (H + L), and Texas Red Affini Pure
Goat anti mouse, IgG (H + L), respectively. These reagents were
provided by Jackson Immuno Research Laboratories (West
Grove, PA, USA). The staining of two types of antibodies were
used against livin and GAPDH, made on the same slide.
Primary antibodies (livin and GAPDH) were diluted to its
optimal dilution (1/100) in diluents. Then, 50 μl of the primary
antibodies was added to the slides and incubated for 45 min at
37°C before rinsing slides with PBS. The following steps must
be performed in darkness.
The secondary conjugated antibodies were diluted to its optimal
dilution (1/50) in diluents. Thereafter, 50 μl of secondary
antibody was added to each slide; these slides were then
incubated for 1 h at 37°C before washing in PBS (2 × 10 min).
In this study, livin and GAPDH were stained with FITC and
Texas Red conjugation, respectively. Finally, all slides were
covered with a cover slip, sealed with clear nail polish, and
kept in a cold and dark place.
Image analysis
Prepared slides were examined using LEICA fluorescence
microscope (BZ00) (LEICA, Germany) with filters set
appropriately for FITC and Texas Red dyes (Chroma, USA).
Two images were captured from each microscope field, one
with blue filter (350–450 nm) and the other with green filter
(550–650 nm).
Furthermore, 20–30 images were captured for each sample
from different area of the tissue section randomly. Images
were captured using a cooled charge‑coupled device
camera (LEICA: DC 350F) (Wetzlar, Germany) interfaced
with a PC. We analyzed these captured images using image
processing algorithms in MATLAB 7 software (http://www.
mathworks.com).
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Ziaei, et al.: Livin expression in Hodgkin lymphoma
The whole boundary of desired cells in both green and red
planes was selected from our available images and then the
ratio of intensity between the mean of pixels in the green and
red plane of selected cells was computed by following ratio
formula:
Mean of pixels intensity for a cell in the
Ratio =
green plane ( indicated livin expression )
Mean of pixels intensity for the same cell in the red
plane ( indicated GAPDH expression )
Unlike some previous studies,[25,31] samples were analyzed one by
one to acquire highly accurate and trustworthy results. Among all
captured images for every sample, 30 cell‑collections were selected
and analyzed by MATLAB 7 (http://www.mathworks.com).
We excluded fluorescence artifacts from the evaluation by
selecting appropriate cells. Typical lymphocytes were our desired
cells for analysis. We selected and confirmed 20 morphologically
definite typical neoplastic HRS cells in HL samples.
Statistical analysis
One‑way analysis of variance and Student’s t‑test were run to
analyze the mean level of livin expression ratio between case
and control groups and their subdivisions. In addition, χ2 and
Fisher’s exact tests were applied to analyze data in clinical
features of this study [Tables 1 and 2].
Results
Among 39 HL cases, patient number 10, 9, 4, and 8 were
diagnosed to be at stages I, II, III, and IV, respectively. The
total cell count was around 2060 cells in all prepared HL and
control sections.
To determine livin upregulation in neoplastic tissues, 1030
infiltrating cells were analyzed in the tumor environment
and approximately the same numbers of background cells
in normal and reactive environment of lymph node were
considered. Mean ratio (M.R) of livin/GAPDH expression in all
HL cases was 0.5459 ± 0.0612 and, in the control group, it was
equivalent to 0.508 ± 0.0267. Accordingly, livin expression was
significantly higher in HL than normal and reactive samples
(P < 0.001, t‑test) [Figure 1].
Surprisingly, no statistical difference between the livin expression
in normal (M.R of livin/GAPDH expression = 0.5085 ± 0.0284)
and reactive hyperplastic lymph nodes (M.R of
livin/GAPDH expression = 0.5068 ± 0.0213) (P > 0.05) was
not observed [Figure 2].
In addition, all diagnosed malignant cells (HRS) in HL [Figure 3]
were analyzed for livin expression (M.R of livin/GAPDH
expression = 0.5902 ± 0.0135) and compared with all infiltrating
cells (M.R of livin/GAPDH expression = 0.5459 ± 0.06126)
in HL, (P < 0.001, paired t‑test). The expression of livin
in malignant cells was significantly higher than reactive
inflammatory cells either (P < 0.001, paired t‑test) [Figure 4].
Cases number 19 and 13 were recognized as early‑stage (I and
II) and late‑stage tumors (III and IV), respectively. In the first
Oncobiology and Targets - Vol 3 | 2016
group, M.R of livin/GAPDH expression was 0.5288 ± 0.0418
and, for the second one, it was 0.5801 ± 0.06941. The differences
between stages in HL were statistically significant (P < 0.01,
t‑test) [Figures 2 and 5].
Based on varying proportion of neoplastic and nonneoplastic
cells between 39 HL samples, 18, 14, 4, 2, and 1 cases were
diagnosed with NHCHL, MCCHL, LRCHL, LDCHL, and
NLPHL histological patterns, respectively. There was no
significant relationship between livin expressions in different
entities of HL [Figures 3 and 6]. Although the pattern of livin
expression is the same for all histological types of Hodgkin
lymphoma, it may be acceptable for NHCHL and MCCHL.
However, there are a few number of other types such as
LRCHL, LDCHL, and NLPHL [Figure 6].
For difficult cases which area under the cureve (AUC) of 0.761,
the receiver operating characteristic (ROC) curve determine
Table 1: Clinical features of patients with Hodgkin
lymphoma
No. of cases
Sex
Male
Female
Age (years)
B symptom
Positive
Fever
Night sweat
Weight loss
Negative
Lymph node involvement
Cervical
Axillary
Mediastinal
Para-aortic
Perihilar
Inguinal
Extra-lymphatic organ involvement
Lung
Liver
Spleen
Bone Marrow
Others
21
17
33.6±15.1
14
9 out of 14
7 out of 14
8 out of 14
17
26
5
9
3
3
4
3
2
3
0
4
Table 2: The cut‑off points to discriminate Hodgkin’s
lymphoma from non neoplastic lymph node according
to mean ratio of livin/glyceraldehyde‑3‑phosphate
dehydrogenase expression
Optimal Cut-off
point
Cut-off point with
higher sensitivity
Cut-off point with
higher specificity
Points Sensitivity Specificity
AUC
%
%
0.5231
74.9
74.1
0.761 (95% CI
0.648 to 0.874)
0.5032
82.1
35.9
0.761 (95% CI
0.648 to 0.874)
0.5334
61.5
89.7
0.761 (95% CI
0.648 to 0.874)
AUC: Areas Under the Curve, 95% CI: 95% confidence interval
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Ziaei, et al.: Livin expression in Hodgkin lymphoma
Discussion
the optimal cut‑off point for mean ratio of livin/GAPDH
expression at 0.5231. This maximizes true positives and true
negatives (sensitivity 74.9%, specificity 74.4%) which enabled
us to distinguish HL from nonneoplastic lymph node samples
as control group [Figure 7] in difficult cases with AUC of
0.761 [Figure 4 and Table 2].
This study revealed prognostic significance of livin protein in
HL. Livin expression in some human malignancies described
before[13,32] as well as malignant melanoma that is illustrated
as melanoma IAP.[21] Since upregulation and/or abnormal
livin expression is proved in tumor genesis and progression
of cancer,[17] over expression of livin is illustrated as a potential
prognostic and diagnostic biomarker. [21] The prognostic
importance of livin has explained in neuroblastoma, bladder
cancer, and Non‑Hodgkin’s Lymphoma (NHL).[21,28,33,34]
The ROC curve revealed that the mean ratio of livin/GAPDH
expression could be an accurate indicator to distinguish HL
from control group (AUC = 0.761, P < 0.0001). Clinical features
of case groups are shown in Table 1.
Figure 1: Livin/glyceraldehyde 3‑phosphate dehydrogenase expression in 39
Hodgkin’s lymphoma patients and 39 controls (normal and reactive) lymph nodes.
The expression of livin protein in Hodgkin’s lymphoma and control groups was
significant statistically (P < 0.001)
a
Figure 2: Livin/glyceraldehyde 3‑phosphate dehydrogenase expression
in 30 normal and nine ones with reactive hyperplasia lymph nodes. The livin protein
expression between these groups was not significant statistically
b
c
d
Figure 3: Glyceraldehyde 3‑phosphate dehydrogenase expression detected by fluorescein and Texas Red conjugated antibodies, respectively (a and b) are related to
infiltrating reactive cells in Hodgkin’s lymphoma. Parts (c and d) show Hodgkin and Reed‑Sternberg cells in Hodgkin’s lymphoma
4
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Ziaei, et al.: Livin expression in Hodgkin lymphoma
Figure 4: Livin/glyceraldehyde 3‑phosphate dehydrogenase expression in
malignant (Hodgkin and Reed‑Sternberg) and nonmalignant cells in Hodgkin’s
lymphoma. Quantification of livin expression between these different populations
was significant (P < 0.001)
Figure 6: The mean ratio of livin/glyceraldehyde 3‑phosphate dehydrogenase
expression among five histological entities of Hodgkin’s lymphoma. No significant
relevance is noticed between livin expressions
Gazzaniga et al.[34] explained that livin can be utilized as
a marker of early recurrence. They proved that median
relapse‑free time of livin‑positive patients is shorter than
livin‑negative patients. Subsequently, Kim et al.[21,33] had shown
positive correlation of livin with prognosis and outcome of
the disease in neuroblastoma cells. Gao et al.[28] found that
expression of livin mRNA and protein was positively correlated
with clinical stage of NHL, B symptom, and some other clinical
features.
Figure 5: Comparison of livin/glyceraldehyde 3‑phosphate dehydrogenase
expression between Early‑stage and advanced‑stage groups of Hodgkin’s
lymphoma patients. Alteration of livin expression was statistically significant in
different clinical stages of Hodgkin’s lymphoma (P < 0.001)
Figure 7: Diagnostic statistics = Receiver operating characteristic curve for
livin/glyceraldehyde 3‑phosphate dehydrogenase expression in discriminating
classical Hodgkin’s lymphoma from nonneoplastic lymph node samples as control
group (area under the cureve = 0.761)
livin in both neoplastic and nonneoplastic cell population are
statistically significant regarding the cell population presenting
in the control lymph nodes. Surprisingly, we found that
livin expression in malignant cells is elevated regarding the
nonneoplastic background cells. As a result, increased livin
expression leads to decreased apoptosis and tumorigenesis.
In contrast, there are some studies on Lung and colon
cancers, [35] colon cancer, [27] and metastatic melanoma,
persisting on correlation between livin overexpression and
clinical outcome despite the up‑regulation of livin in these
malignancies.
Considering the role of nuclear factor‑kappa B (NFκB) on
up‑regulation of IAPs,[36] it is interpreted that TF (NFκB) is
activated in the reprogrammed HRS cells.[2] This activation can
be effective in upregulation of IAPs (livin is one of them) in the
malignant cells compared with inflammatory background cells.
More comprehensive molecular profiling studies are required
to underpin this issue.
The present study is the first description of evaluation and
clinical significance of livin expression in HL. We evaluated
livin expression not only in the bulk of infiltrating background
cells but also in malignant ones and then compared livin
expression in involved lymph nodes to nonneoplastic
lymphoid tissues. We found that differences in expression of
Our findings revealed that difference in livin expression
between the early stage cases and advanced‑stage HL samples
may be significant. In addition, our results unveiled no
correlation between the level of the livin expression and other
factors such as age, gender, B symptom, and lymphoid and
extra‑lymphoid tissues involvements.
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Ziaei, et al.: Livin expression in Hodgkin lymphoma
The present study proposed that livin might be considered a
prognostic factor for determination of stages in HL as well as
to be targeted in treatment of HL patients.
In the present study, LDCHL samples (the most invasive form
of HL[4]) had the lowest M.R of livin/GAPDH expression while
LRCHL cases (with a rich background of normal lymphocytes)
had the highest M.R. of livin/GAPDH expression [Figure 6].
This might be attributed to the restricted numbers of samples
which were studied here. Because the expression of GAPDH
is constant in normal and neoplastic lymphoid tissues, any
differences in livin/GAPDH ratio indicate changes in livin
protein expression.[37]
Hence, the authors recommended more sophisticated studies
with more expanded samples of HL. It is concluded that livin
over expression is significant in pathogenesis and progression
of HL. It may become an attractive candidate for targeted
therapies through different strategies as listed elsewhere.[21]
Nevertheless, it is unidentified whether pan‑IAP inhibitors or
specific livin inhibitors are the most useful therapeutically for
treatment of malignancies. Evaluation of all members of IAP
family is, therefore, recommended to find a more vivid picture
of their role in different stages of HL. We have extended our
study to evaluate NAIP expression profile in HL.[37]
Acknowledgments
This research work was supported by Grant No. 187070
from the Deputy for Research, Isfahan University of
Medical Sciences, Isfahan, Iran. We would like to appreciate
Dr. Ardeshir Talebi (Department of Pathology, Isfahan
University of Medical Sciences) for pathology diagnosis of
cases, Dr. Mohammad Reza Mohajeri and Ms. Sahar Mahmoodi
for sample preparation and immune staining of samples.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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