Ki-67 expression is predictive of prognosis in patients with stage I/II extranodal NK/T-cell lymphoma, nasal type
Ki-67 expression is predictive of prognosis in patients with stage I/II extranodal NK/T-cell lymphoma, nasal type
S. J. Kim 2
B. S. Kim 2
C. W. Choi 2
J. Choi 1
I. Kim 1
Y.-H. Lee 0
J. S. Kim 2
0 Department of Diagnostic Radiology, Korea University Medical Center , Seoul , Korea
1 Department of Pathology
2 Department of Internal Medicine
Background: Localized extranodal natural killer (NK)/T-cell lymphoma, nasal type, commonly has a low or low-intermediate risk of the international prognostic index (IPI), so the IPI has shown inconsistency in predicting prognosis. Thus, we analyzed Ki-67 expression and proposed a new prognostic model including Ki-67 expression for stage I/II extranodal NK/T-cell lymphoma. Patients and methods: We studied Ki-67 expression and its relationship with prognosis in 50 patients with extranodal NK/T-cell lymphoma. Results: The patients were dichotomized by the median value: low (<65%) versus high Ki-67 (‡65%). High Ki-67 was associated with a worse overall survival (OS; P = 0.021) and disease-free survival (DFS; P = 0.044). In multivariate analysis, Ki-67 expression and primary site of involvement were found to be an independent prognostic factor for OS and DFS (P < 0.05). Based on these results, we proposed a new clinico-pathological prognostic model with Ki-67 expression and the primary site of involvement. It showed a high degree of correlation with worse OS and DFS (P < 0.001). Conclusions: Ki-67 expression is predictive of prognosis, and our prognostic model may become a useful tool for predicting prognosis in patients with stage I/II extranodal NK/T-cell lymphoma, nasal type.
Extranodal natural killer (NK)/T-cell lymphoma, nasal type,
is a distinct subtype of non-Hodgkin’s lymphoma that is very
rare in North America and Europe [
]. However, it is rather
common in Asia and South America . Thus, it has been
reported to account for 8.7% of all non-Hodgkin’s lymphomas
and 74.1% of lymphomas that develop in the nasal cavity and
paranasal sinuses in Korea [
]. Pathologically, extranodal
NK/T-cell lymphoma has unique characteristics including the
expression of cytoplasmic CD3, CD56 and cytotoxic molecules
such as TIA-1, and is positive for Epstein–Barr virus (EBV)
in situ hybridization [
]. Clinically, it frequently occurs in
middle-aged men, and usually presents as a localized disease
involving the head and neck. Furthermore, most patients
show good performance status, thus most patients have low
international prognostic index (IPI) scores [
]. However, the
overall prognosis of this disease is poor because of frequent
relapse or resistance to treatment [
]. This discrepancy
has led to conflicting results about the prognostic value of
the IPI score in extranodal NK/T-cell lymphoma, nasal type.
Thus, although a positive correlation of IPI with prognosis
was reported in this rare subtype of non-Hodgkin’s
lymphoma , other studies have not shown a correlation
of IPI with prognosis in extranodal NK/T-cell lymphoma
]. Because the IPI mainly depends on the clinical
characteristics, another prognostic indicator reflecting
biological characteristics, such as a rapid growth of tumor
cells, may help to predict differences in survival among
patients with localized extranodal NK/T-cell lymphoma
showing low IPI scores.
Ki-67 is a nuclear antigen expressed by dividing cells.
Thus, the percentage of Ki-67-positive cells reflects the
proportion of actively proliferating tumor cells. Although
the prognostic value of Ki-67 remains controversial in
non-Hodgkin’s lymphoma, a recent study in peripheral T-cell
lymphoma showed a positive correlation with poor prognosis
]. However, the prognostic value of Ki-67 has never been
studied in patients with extranodal NK/T-cell lymphoma.
Therefore, we performed this study to determine the value of
Ki-67 expression in predicting prognosis in patients with
localized extranodal NK/T-cell lymphoma.
materials and methods
Fifty patients diagnosed with localized extranodal NK/T-cell lymphoma
at the Korea University Medical Center were included from January 2000
to October 2006. They were consecutively diagnosed from 2000, and
had paraffin-embedded blocks available for Ki-67 immunohistochemical
staining. Patients were diagnosed based on the following criteria:
the presence of a tumor mass with histological features and
immunophenotypes compatible with NK/T-cell lymphoma (cCD3+,
CD20–, CD56+, cytotoxic molecules +, EBV in situ hybridization +). All
patients had undergone staging investigations including: complete blood
counts, serum biochemistry, serum lactate dehydrogenase (LDH), serum
b2 microglobulin, bone marrow aspiration and trephine biopsy, endoscopic
examination of the nasal and oral cavity, computed tomographic (CT)
or magnetic resonance imaging (MRI) of the involved organs of the head
and neck and CT of the chest and abdomen-pelvis. The Ann Arbor staging
system was used; however, the size of the primary lesion was calculated
by the sum of products of the longest diameters and greatest perpendicular
diameters to define the bulk of the lesion (‡10 cm2). The reason why the
sum of products was used instead of diameter, e.g. mass with >10 cm
diameter, was that most cases occurred in the head and neck, such as
paranasal sinuses, thus the size of the primary lesion was commonly smaller
than other types of non-Hodgkin’s lymphoma.
Treatment modalities that patients received were as follows:
chemotherapy followed by involved-field radiotherapy (19 patients) and
chemotherapy alone (31 patients). CHOP (cyclophosphamide, doxorubicin
hydrochloride, vincristine, prednisolone)-like chemotherapy regimens were
used including CEOP-B (cyclophosphamide, epirubicin, vincristine,
prednisolone and bleomycin), and CEOP-E (cyclophosphamide, epirubicin,
vincristine, prednisolone and etoposide). Involved-field radiotherapy of
44–60 Gy was delivered in daily fractions of 1.8–2.0 Gy by a conventional
fractionation schedule (five fractions per week).
immunohistochemistry for Ki-67. For the detection of Ki-67, we used the
anti-Ki-67 mouse monoclonal antibody (clone MIB-1, DakoCytomation,
Carpinteria, CA, USA). First, tissue sections were dewaxed and rehydrated.
After endogenous peroxidase was quenched with methanol and 3% hydrogen
peroxide for 5 min, antigen retrieval was done with citrate buffer (pH 6.0)
in a pressure cooker for 2 min. After blocking with 10% donkey serum, the
slides were incubated with the primary antibody (dilution 1:100) for 30 min
at room temperature, and washed with Tris-buffered saline (TBS). The
secondary antibody (ChemMate Dako Envision, DakoCytomation) was
applied for 30 min. Slides were again washed with TBS and color was
developed by 5-min incubation in diaminobezidine (DAB) solution. Slides
were counterstained with hematoxylin. Tumor cells were considered
positive for the Ki-67 antigen if there was intranuclear DAB staining.
After the slides were scanned at low magnification (·40), the cells with
positively stained nuclei were counted at high magnification (·400).
The percentage of Ki-67 expression was quantified by determining the
number of positive cells expressing nuclear Ki-67 among the total
number of tumor cells in the high-power field. The median percentage
(65%) of Ki-67 expression was designated as a cutoff, thus cases higher
than the median value (‡65%) was defined as high Ki-67 expression, and
less than the median value (<65%) was designated as low Ki-67 expression
for the survival analysis. Slides were reviewed by pathologists (J.C. and I.K),
and all interpretations of immunohistochemistry were performed without
knowledge of clinical outcome.
statistical analysis. The relationship of Ki-67 expression with clinical
variables was evaluated using the chi-squared test. The period of overall
survival (OS) was measured from the date of diagnosis to the date of death
or the last follow-up visit. The disease-free survival (DFS) was measured
from the date treatment began to the date when disease progression was
recognized or the date of the last follow-up visit. The Kaplan–Meier
method was used to calculate OS and DFS, and survival curves were
compared by the log-rank test. The Cox proportional hazards regression
model was used for the multivariate analysis to compare the factors
proven significant in the univariate analysis. A two-sided P-value of less
than 0.05 was considered statistically significant.
characteristics of patients
The clinical characteristics of the 50 patients are presented in
Table 1. Thirty patients (60.0%) initially presented as stage I
and 20 patients as stage II. In 11 patients, their primary
lesion was bulky (‡10 cm2). The number of patients with
extranodal NK/T-cell lymphoma originating from the nasal
cavity and nasopharynx was 33 (66.0%). Seventeen patients
(34.0%) presented with lymphomas from the upper
aerodigestive tract: tonsil (n = 10), oropharynx (n = 5),
hypopharynx (n = 1), palate (n = 1).
response to treatment and outcomes. The median OS was
42.43 months (95% confidence interval (CI), 13.45–71.42)
and the estimated 5-year OS rate was 43.9%. Twenty-three
patients (46.0%) and 16 patients achieved complete
remission (CR) and partial response (PR), respectively, thus
the overall response rate was 78.0%. Among them, 28 patients
showed evidence of relapse or disease progression after
achieving CR or PR. The median DFS was 19.03 months
(95% CI, 5.89–32.17). When the survival difference was
analyzed based on the type of treatment, chemotherapy
followed by radiotherapy failed to show a significant
benefit over chemotherapy alone in terms of OS and DFS
(P = 0.653, and 0.467, respectively). This is the same as the
results of our previous report [
Ki-67 expression. The nuclear staining pattern of Ki-67
expression was evaluated and the quantified Ki-67 expression
(the percentage of Ki-67-positive cells to tumor cells) was noted
to have a wide range of distribution from 5% to 95%
(Figure 1). When the patients were separated into two
groups based on the median value (65%), high Ki-67 (‡65%)
and low Ki-67 (<65%), its relationship with clinical features is
summarized in Table 2. Bulky primary lesions was significantly
associated with high Ki-67 expression (P = 0.006), suggesting
that bulky tumors may have a high proliferation of tumor
cells. However, there was no association of Ki-67 expression
with other clinical features such as stage, IPI, B symptoms
and so forth except for Eastern Cooperative Oncology Group
(ECOG) performance status. The group with high Ki-67
showed increased cases with elevated serum LDH and b2
microglobulin compared with the low Ki-67 group; however,
it failed to show a statistical significance (P = 0.112 and 0.098,
respectively). The status of Ki-67 expression was not
different between the chemotherapy and chemotherapy plus
radiotherapy groups (P = 0.773). The response to
chemotherapy was not significantly associated with the status
of Ki-67 expression (P > 0.05). The mean percentage of Ki-67
expression in patients responding to chemotherapy was not
significantly different from non-responders (54.74 6 26.82 vs.
58.63 6 27.84).
survival analysis. In the univariate analysis, the group with high
Ki-67 expression had a shorter OS (P = 0.021, Figure 2A) and
DFS (P = 0.044, Figure 2B). The results of the univariate
analysis are summarized in Table 3. The primary site of
involvement was significantly associated with a poor OS
(P = 0.022), and this was consistent with our previous
]. Thus, NK/T-cell lymphomas in the nasal cavity/
nasopharynx were associated with worse OS. Bulky tumors
(‡10 cm2) showed a marginal significance with OS (P = 0.052).
However, the IPI failed to show prognostic significance for
OS (P = 0.296). The association of DFS with IPI, bulky tumors,
B symptoms and primary site of involvement did not reach
statistical significance (P > 0.05). Serum LDH was found to
be significantly associated with DFS (P = 0.045). Ann Arbor
stage failed to discriminate a survival difference between
stages I and II (P > 0.05). In the multivariate analysis with
the parameters used in the univariate analysis, Ki-67
expression was found to be an independent prognostic
factor for OS (response rates (RR) = 4.330; 95% CI, 1.108–
16.918; P = 0.035) and DFS (RR = 3.230, 95% CI 1.073-9.720,
P = 0.036). The primary site of involvement was also
an independent prognostic factor for OS (RR = 6.105;
95% CI, 1.530–24.363; P = 0.010) and DFS (RR = 5.028;
95% CI, 1.514–16.698; P = 0.008).
prognostic model with Ki-67 expression and primary site of
involvement. According to the results of our univariate and
multivariate analyses, we proposed a new prognostic model
with two poor prognostic factors: Ki-67 expression and primary
site of involvement. Thus, we designated a high-risk group as
the presence of two factors including high Ki-67 expression
and nasal cavity/nasopharynx-originated NK/T-cell
lymphomas and a low-risk group as the presence of one
factor or none. This new prognostic model showed a high
degree of correlation with survival outcomes in terms of OS
and DFS (P < 0.001; Figure 3A, B).
Since the IPI was proposed by the International Non-Hodgkin’s
Lymphoma Prognostic Factors Project, the IPI has been used
for both predicting prognosis and selecting therapeutic options
in patients with aggressive non-Hodgkin’s lymphoma.
However, its value has been challenged by extranodal NK/T-cell
lymphoma because it has failed to predict prognosis in
retrospective analyses [
]. Thus, newer prognostic models
have been suggested to supplement the IPI for extranodal
NK/T-cell lymphoma [
]. However, these prognostic
models are based primarily on pre-treatment clinical
Volume 18 | No. 8 | August 2007
characteristics; the molecular factors that may predict the
aggressiveness and malignant potential of extranodal NK/T-cell
lymphoma have not yet been defined.
The status of cellular proliferation and cell-cycle regulation
have been studied for possible molecular prognostic markers
in non-Hodgkin’s lymphoma including cell-cycle regulatory
molecules TP53, p27kip1 and cyclin D [
]. The uptake of
fluorodeoxyglucose (FDG) in positron emission tomography
(PET) is known to be related to cellular proliferation and
has been reported to predict prognosis in patients with
non-Hodgkin’s lymphoma . Recently, Ki-67 proliferation
rate was also reported to be correlated with increased
angiogenesis in the canine non-Hodgkin’s lymphoma
spontaneous model, suggesting its relationship with
aggressive behavior of lymphomas [
measurement of the proliferation status before
commencement of treatment may improve the overall
prognostic assessment of patients with non-Hodgkin’s
lymphoma allowing therapy to be tailored to patients.
The measurement of Ki-67 expression using
immunohistochemistry techniques is one of the methods
available to measure cellular proliferation. Ki-67 is a protein
synthesized as a cell begins proliferation; thus it is present in the
proliferation-associated phase of the cell cycle including S, G2
and M [
]. Immunohistochemical staining of cells with Ki-67
monoclonal antibody (clone MIB-1) is a commonly used
technique that can be more easily applied to formalin-fixed,
paraffin-embedded tissue sections compared with other
methods such as mitotic figure counting and measurement of
proliferating cell nuclear antigen (PCNA) [
]. This technique
allows for the differentiation of proliferating cells from
quiescent cells. Thus, the proliferation index, which is the
percentage of Ki-67-positive cells among the total tumor cells
in a selected area, has been used to assess the proliferative
activity of tumors. Ki-67 expression has been reported to be
associated with tumor aggressiveness and poor prognosis in
a variety of malignancies [
The prognostic significance of Ki-67 has been studied in
several subtypes of non-Hodgkin’s lymphoma. Previous
retrospective analyses of diffuse large B-cell lymphoma, have
reported that the proportion of proliferating cells, marked by
Ki-67 expression, correlated with poor treatment outcome and
]. A recent retrospective analysis of peripheral
T-cell lymphoma has shown the prognostic significance of
Ki-67 expression; the results of this study suggested
a prognostic model based on age (>60 years), high LDH, poor
performance status and high Ki-67 expression . However,
a study by the Nordic Lymphoma Group showed that the
expression of Ki-67 was not associated with survival difference
in patients with diffuse large B-cell lymphoma [
the reason for these inconsistent results regarding the
prognostic significance of Ki-67 expression in non-Hodgkin’s
lymphoma remains unclear, the following might be possible
explanations. First, a different definition for high Ki-67
expression might be related to different results. The arbitrarily
defined various cut-offs from 20% to 80% were used for high
Ki-67 expression to dichotomize their study populations into
high and low Ki-67 expression [
]. Second, lymphoma
cells having high proliferative activity, represented by Ki-67
expression, may be more sensitive to chemotherapy than cells
with low Ki-67 expression. However, our study used the
median value to define high Ki-67 expression so that we could
avoid the first problem. Furthermore, the response to treatment
was no different depending on the status of Ki-67 expression,
thus the second problem may not be applicable to our results.
In this study, high Ki-67 expression defined by the median
value (65%) was significantly associated with a shorter OS
and DFS (P < 0.05), while Ann Arbor stage and IPI failed to
predict prognosis of the patients with extranodal NK/T-cell
lymphoma. We also found a relationship between high Ki-67
expression and bulky tumors (‡10 cm2). These findings
suggest that the extent of Ki-67 expression may reflect the
proliferative activity of NK/T-cell lymphoma. In our study,
a new definition of bulky tumors was used with the sum of
products of the longest diameters and greatest perpendicular
diameters (‡10 cm2). Considering that most cases of extranodal
NK/T-cell lymphoma occurred in the head and neck, such as
in the paranasal sinuses, a previous definition of bulky
disease (a lesion with >10 cm diameter) can rarely be applied
to this disease entity. In fact, there were no cases having
a lesion of >10 cm diameter in our study.
Because the patient population of this study was confined to
localized disease (stage I and II) with low or low–intermediate
IPI risk, our results support the idea that cellular
proliferation, represented by Ki-67 expression, can identify
patients with extranodal NK/T-cell lymphoma that may show
aggressive clinical course and poor treatment outcome. Thus,
pre-treatment evaluation of Ki-67 expression may become
a marker for predicting prognosis and rendering a more
tailored treatment strategy in patients with extranodal
NK/T-cell lymphoma. Based on our results, we propose
a new prognostic model with high Ki-67 expression and the
primary site of involvement. Because high Ki-67 expression
showed a significant relationship with the size of primary
tumors, this model including Ki-67 expression can reflect the
aggressive biological characteristics of extranodal NK/T-cell
lymphoma. Furthermore, its prognostic significance can be
augmented by the fact that nasal cavity/nasopharynx-originated
NK/T-cell lymphomas showed inferior survival outcome
compared with upper aerodigestive tract lymphomas. Thus,
this new prognostic model may be a useful prognostic index
for localized extranodal NK/T-cell lymphoma, nasal type.
In conclusion, this study demonstrated the prognostic
significance of Ki-67 expression in extranodal NK/T-cell
lymphoma, especially localized disease with low and
low–intermediate IPI risk. Considering the limited role of the
Ann Arbor stage and IPI in extranodal NK/T-cell lymphoma,
the assessment of Ki-67 expression and our new prognostic
model may become a useful tool for predicting prognosis
and deciding upon a therapeutic strategy in patients with
localized extranodal NK/T-cell lymphoma, nasal type.
The Brain Korea 21 project supported this work.
1. Jaffe ES , Chan JK , Su IJ et al. Report of the Workshop on Nasal and Related Extranodal Angiocentric T/Natural Killer Cell Lymphomas . Definitions, differential diagnosis, and epidemiology . Am J Surg Pathol 1996 ; 20 : 103 - 111 .
2. Harris NL , Jaffe ES , Diebold J et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-Airlie House , Virginia November 1997 . J Clin Oncol 1999 ; 17 : 3835 - 3849 .
3. Aozasa K , Ohsawa M , Tajima K et al. Nation-wide study of lethal mid-line granuloma in Japan: frequencies of Wegener's granulomatosis, polymorphic reticulosis, malignant lymphoma and other related conditions . Int J Cancer 1989 ; 44 : 63 - 66 .
4. Ko YH , Kim CW , Park CS et al. REAL classification of malignant lymphomas in the Republic of Korea: incidence of recently recognized entities and changes in clinicopathologic features . Hematolymphoreticular Study Group of the Korean Society of Pathologists. Revised European-American lymphoma. Cancer 1998 ; 83 : 806 - 812 .
5. Kanavaros P , Lescs MC , Briere J et al. Nasal T-cell lymphoma: a clinicopathologic entity associated with peculiar phenotype and with EpsteinBarr virus . Blood 1993 ; 81 : 2688 - 2695 .
6. Lee J , Suh C , Park YH et al. Extranodal natural killer T-cell lymphoma, nasaltype: a prognostic model from a retrospective multicenter study . J Clin Oncol 2006 ; 24 : 612 - 618 .
7. Kim TM , Park YH , Lee SY et al. Local tumor invasiveness is more predictive of survival than International Prognostic Index in stage I(E)/II(E) extranodal NK/T-cell lymphoma, nasal type . Blood 2005 ; 106 : 3785 - 3790 .
8. Kim GE , Cho JH , Yang WI et al. Angiocentric lymphoma of the head and neck: patterns of systemic failure after radiation treatment . J Clin Oncol 2000 ; 18 : 54 - 63 .
9. Lee J , Park YH , Kim WS et al. Extranodal nasal type NK/T-cell lymphoma: elucidating clinical prognostic factors for risk-based stratification of therapy . Eur J Cancer 2005 ; 41 : 1402 - 1408 .
10. Chim CS , Ma SY , Au WY et al. Primary nasal natural killer cell lymphoma: long-term treatment outcome and relationship with the International Prognostic Index . Blood 2004 ; 103 : 216 - 221 .
11. Cheung MM , Chan JK , Lau WH et al. Early stage nasal NK/T-cell lymphoma: clinical outcome, prognostic factors, and the effect of treatment modality . Int J Radiat Oncol Biol Phys 2002 ; 54 : 182 - 190 .
12. Aviles A , Diaz NR , Neri N et al. Angiocentric nasal T/natural killer cell lymphoma: a single centre study of prognostic factors in 108 patients . Clin Lab Haematol 2000 ; 22 : 215 - 220 .
13. Went P , Agostinelli C , Gallamini A et al. Marker expression in peripheral T-cell lymphoma: a proposed clinical-pathologic prognostic score . J Clin Oncol 2006 ; 24 : 2472 - 2479 .
14. Kim SJ , Kim BS , Choi CW et al. Treatment outcome of front-line systemic chemotherapy for localized extranodal NK/T cell lymphoma in nasal and upper aerodigestive tract . Leukemia Lymphoma 2006 ; 47 : 1265 - 1273 .
15. Saez A , Sanchez E , Sanchez-Beato M et al. p27KIP1 is abnormally expressed in diffuse large B-cell lymphomas and is associated with an adverse clinical outcome . Br J Cancer 1999 ; 80 : 1427 - 1434 .
16. Filipits M , Jaeger U , Pohl G et al. Cyclin D3 is a predictive and prognostic factor in diffuse large B-cell lymphoma . Clin Cancer Res 2002 ; 8 : 729 - 733 .
17. Lapela M , Leskinen S , Minn HR et al. Increased glucose metabolism in untreated non-Hodgkin's lymphoma: a study with positron emission tomography and fluorine-18-fluorodeoxyglucose . Blood 1995 ; 86 : 3522 - 3527 .
18. Patruno R , Zizzo N , Zito AF et al. Microvascular density and endothelial area correlate with Ki-67 proliferative rate in the canine non-Hodgkin's lymphoma spontaneous model . Leukemia Lymphoma 2006 ; 47 : 1138 - 1143 .
19. Schluter C , Duchrow M , Wohlenberg C et al. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins . J Cell Biol 1993 ; 123 : 513 - 522 .
20. Cattoretti G , Becker MH , Key G et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections . J Pathol 1992 ; 168 : 357 - 363 .
21. Scholzen T , Gerdes J. The Ki- 67 protein: from the known and the unknown . J Cell Physiol 2000 ; 182 : 311 - 322 .
22. Grogan TM , Lippman SM , Spier CM et al. Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67. Blood 1988 ; 71 : 1157 - 1160 .
23. Hall PA , Richards MA , Gregory WM et al. The prognostic value of Ki67 immunostaining in non-Hodgkin's lymphoma . J Pathol 1988 ; 154 : 223 - 235 .
24. Jerkeman M , Anderson H , Dictor M et al. Assessment of biological prognostic factors provides clinically relevant information in patients with diffuse large B-cell lymphoma-a Nordic Lymphoma Group study . Ann Hematol 2004 ; 83 : 414 - 419 .
25. Miller TP , Grogan TM , Dahlberg S et al. Prognostic significance of the Ki-67-associated proliferative antigen in aggressive non-Hodgkin's lymphomas: a prospective Southwest Oncology Group trial . Blood 1994 ; 83 : 1460 - 1466 .