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Introduction
Non-Hodgkin's lymphoma (NHL) forms a highly heterogeneous group of diseases. Despite steady improvement in their
classification, patients with the same diagnosis can have markedly different clinical courses. Since prognosis remains
uncertain, several biologically-based prognostic indicators have been proposed in addition to the international prognostic
index (IPI) scores. However, none have proven to be completely
effective[1], so novel independent factors are required to
better characterize the malignant potential of NHL.
Accumulating evidence suggests that abnormalities in cell cycle and apoptosis are hallmarks of clonal expansion leading
to cancer emergence[2,3]. Polo-like kinase 1 (PLK1) has been implicated in various aspects of the progression of the cell
cycle[4], including centrosome
maturation[5], assembly of the mitotic
spindle[6], regulation of the anaphase-promoting
complex[7], and
cytokinesis[8]. Survivin, a new inhibitor of the apoptosis protein (IAP) family, directly inhibits caspase 3 and caspase 7
and is one of the strongest anti-apoptosis factors by
far[9]. Although PLK1 and survivin play completely different roles, they
have some characteristics in
common[10_13]: they are mostly expressed in the
G2/M phase of the cell cycle and they are not
expressed in most normal differentiated tissues; however, they are
overexpressed in a broad spectrum of cancer types and the
expression often correlates with poor outcomes. To assess the clinical significance of PLK1 and survivin in NHL, the
expression of PLK1 and survivin were examined and correlated with clinical outcome.
Materials and methods
Patients A total of 88 patients (age: 5_86 years, median 47 years) were diagnosed with NHL at Union Hospital (Wuhan,
China) between January 2001 and June 2006. None of the patients underwent radiotherapy or chemotherapy prior to
diagnosis. Classification was established on standard
hematoxylin-eosin-stained sections according to the guidelines
of the World Health Organization 2001 Classification of
Lymphoid Neoplasms[14]. Eighty-eight cases were treated with
CHOP or E-CHOP or CHOP plus Rituximab (600
mg/m2 CTX, iv, d 1; 50 mg/m2 ADR, iv, d 1; 2
mg/m2 VCR, iv, d 1; 60 mg/m2 Pred, po, d 1_7; 100
mg/m2 etoposide, iv, d 1; 375
mg/m2 Rituximab, iv, d 1). Clinical follow-up data were available for
all patients. The median follow up was 33 months (from 2 to
64 months). As a control for NHL, 5 cases of reactive lymph
node proliferation were included in the study.
Immunohistochemistry on tissue slides Immunostaining
was performed with primary antibodies against PLK1 (1:100;
rabbit polyclonal antibody; Biolegend, San Diego, CA, USA)
and survivin (1:150; rabbit polyclonal antibody; Neomarkers,
Fremont, CA, USA). In brief, 4 µm thick formalin-fixed
paraffin sections were dewaxed and rehydrated through a graded
series of ethanols. After antigen retrieval with microwave
heating, the sections were treated with 0.3% hydrogen
peroxide followed by incubation in bovine serum albumin. The
sections were then incubated with primary antibodies. After
washing with phosphate-buffered saline, the sections were
incubated with biotinylated anti-immunoglobulin G of the
appropriate species and streptavidin peroxidase using a
labeled streptavidin biotin kit (Zhongshan Biotechnology,
Beijing, China). The immunoperoxidase reaction product was
visualized with diaminobenzidine/hydrogen peroxide. The
sections were counterstained with hematoxylin.
Evaluation of tissue staining The staining intensity of
the tissue slides was evaluated independently by 2
pathologists who were blinded to the patients' characteristics and
survival. To assess the differences in staining intensity, an
immunoreactivity scoring system (IRS) was applied. The
intensity of staining was designated as negative (0), weak
(1), moderate (2), or strong (3). Additionally, the percentage
of positive cells was evaluated and scored as either no cells
(0), less then 10% of cells (1), 10%_50% of cells (2),
51%_80% of cells (3), or over 80% of the cells stained (4). By
multiplication of these 2 parameters, the IRS for each case
was calculated. Finally, the cases were grouped as antigen
negative (IRS 0_6) or antigen positive (IRS 7_12) for
statistical analysis[15].
Statistical analysis Patient characteristics were
compared by Fisher's exact test or χ2-test. Generally,
P<0.05 was considered statistically significant. For all statistical
procedures, SPSS v11.5 software (SPSS, Chicago, IL, USA)
was used.
Results
Expression of 2 proteins in NHL In all 88 NHL cases,
63.6% expressed PLK1 and 79.5% expressed survivin. The detailed
distribution is listed in Table 1. The expression of PLK1 and
survivin were then investigated in B-NHL and T-NHL,
respectively. The PLK1 expression rate had no significance
difference between B-NHL and T-NHL (57.8%
vs 77.3%; P=0.103), as did survivin (76.6%
vs 86.4%; P=0.544).
PLK1 was often strongly expressed in nuclei and seldom
weakly in cytoplasm, while survivin was mainly strongly
expressed in nuclei. None of the 5 reactive lymph node
proliferation cases showed sufficient staining of PLK1 or survivin
to be designated as positive (Figures 1, 2).
Relationship between the expression of 2 proteins and
clinical characteristics in B-NHL and T-NHL
In B-NHL, there was a highly significant positive correlation of PLK1
expression with age, Ann Arbor stage, lactate
dehydrogenase (LDH) levels, systemic symptoms, IPI scores, and
therapeutic effect. However, in T-NHL, the PLK1 expression was
only associated with LDH levels, systemic symptoms, and
IPI scores. There were no significant relationships between
the survivin expression and these outcomes in either B-NHL
or T-NHL (Table 2).
Discussion
In recent years, a growing spectrum of biochemical
investigations has contributed to better characterize biological
tumor behavior, thereby improving treatment
strategies[16]. Among these studies, PLK1 and survivin were selected as
the study objects, concerning the 2 important aspects of
tumor biological behavior: proliferation and apoptosis.
PLK1 is a cell cycle-regulated, cyclin-independent
serine/threonine protein kinase, and its protein level changes from
very low level during the G1 phase to at least a 10-fold
increase by the G2/M
phase[17]. PLK1 is involved in mitotic
spindle organization and cytokinesis and seems to play an
essential role in regulating chromosomal
segregation[18]. Furthermore, overexpressed PLK1 can cause the formation
of oncogenic foci in NIH 3T3 cells, which are capable of
forming tumors in nude mice[19]. The elevated expression of
PLK1 had been found in non-small cell lung
cancer[20], squamous cell carcinoma of the head and
neck[21], esophageal
cancer[22], gastric
cancer[23], ovarian
cancer[24], endometrial
cancer[25], colon cancer[26], breast
cancer[27], and malignant
melanoma[28], prostate[29], papillary
carcinoma[30], and oropharyngeal
carcinomas[31]. The expression of PLK1 was
correlated with an adverse clinical outcome in some tumors.
Survivin belongs to the IAP family and it suppresses
apoptosis induced by Fas, Bax, caspases, and anticancer
drugs[32]. Survivin is expressed in a strict cell
cycle-dependent and tissue-restricted manner. It is expressed at high
levels in the G2/M phase and in proliferating
tissues[33]. Overexpressed survivin can be detected in almost all human
tumors and is correlated with poor
prognosis[34].
In the present study, the expression rate of PLK1 was
lower in some less aggressive or indolent types, such as
small lymphocytic lymphoma (SLL) and marginal zone B-cell
lymphoma (MALT) than that in some more aggressive types,
such as diffuse large B-cell lymphoma (DLBCL) and peropheral T-cell lymphoma unspecified (PTLU), while the
expression rate of survivin seemed to have no such trend.
This suggested that PLK1 overexpression was correlated
with cell-proliferating activity, and survivin overexpression
implied the common characterization of NHL: the
dysregulation of apoptosis.
PLK1 overexpression was positively linked to age, Ann
Arbor stage, LDH levels, systemic symptoms, IPI scores,
and therapeutic effect in B-NHL, and was only linked to LDH
levels, systemic symptoms, and IPI scores in T-NHL. The
reason may be the highly heterogeneity and limited number
of cases of T-NHL. There was no significant difference
between the survivin-negative and -positive groups for these
characteristics, either in B-NHL or in T-NHL. It verified again
that apoptosis resistance may be the essence of NHL.
In conclusion, PLK1 and survivin were expressed at high
levels in NHL cases, and PLK1 overexpression was
associated with elderly patient, advanced tumor stage, systemic
symptoms, elevated LDH levels, and poor therapeutic effect.
The overexpression of survivin was not correlated with bad
outcome.
Acknowledgements
The authors thank Dr Xiu NIE and Hua-xiong PAN for
supplying the lymphoma tissues and for their skillful
technical assistance.
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