Expression of Ki-67, cyclin D1 and apoptosis markers correlated with survival in prostate cancer patients treated by radical prostatectomy.
ABSTRACT The study was designed to analyse the prognostic value of proliferation markers Ki-67 and cyclin D1 and apoptosis in prostate cancer (PC) patients treated by radical prostatectomy.
Two hundred and eleven patients treated by radical prostatectomy for localised prostate cancer were clinically followed up for a mean of 7.3 years. The primary histopathological specimens were re-analysed to ensure uniform histoplthological grading and pT classification. A tissue microarray construction (TMA) was used in immunohistochemisty to assess the expression of Ki-67, cyclin D1 and the apoptosis marker Tag. The results were analysed with light microscopy and the findings were compared to standard histology, pT and clinical follow-up data.
The co-expression of Ki-67 and cyclin Dl (p=0.05) was common. High fraction of Ki-67 positive cells and a high fraction of apoptotic cells were often present in same tumours (p=0.05). High apoptotic rate was related to positive surgical margin status (p=0.047). Low expression of Ki-67 was related to a low Gleason score (p<0.001), an absence of either capsule penetration (p = 0.029) or perineural invasion (p=0.004). High expression of cyclin Dl was related to perineural growth (p=0.039). Prostate specific antigen (PSA) recurrence-free survival (RFS) was predicted by Gleason grade (p<0.001) and capsule invasion (p=0.006). High expression of Ki-67 (p=0.03), as well as high apoptotic rate (p=0.04) were related to a high risk of cancer death. In multivariate analysis the seminal vesicle invasion was the only independent predictor of cancer death (p = 0.01).
The expression of Ki-67, cyclin D1 and a high apoptotic rate are related to a malignant phenotype in prostate cancer, but their prognostic value is inferior to standard histological prognostic factors.
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ABSTRACT: The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness.Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 05/2014; · 1.08 Impact Factor
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ABSTRACT: Prostate cancer (PCa) is a potentially curable disease when diagnosed in early stages and subsequently treated with radical prostatectomy (RP). However, a significant proportion of patients tend to relapse early, with the emergence of biochemical failure (BF) as an established precursor of progression to metastatic disease. Several candidate molecular markers have been studied in an effort to enhance the accuracy of existing predictive tools regarding the risk of BF after RP. We studied the immunohistochemical expression of p53, cyclooxygenase-2 (COX-2) and cyclin D1 in a cohort of 70 patients that underwent RP for early stage, hormone naïve PCa, with the aim of prospectively identifying any possible interrelations as well as correlations with known prognostic parameters such as Gleason score, pathological stage and time to prostate-specific antigen (PSA) relapse. We observed a significant (p = 0.003) prognostic role of p53, with high protein expression correlating with shorter time to BF (TTBF) in univariate analysis. Both p53 and COX-2 expression were directly associated with cyclin D1 expression (p = 0.055 and p = 0.050 respectively). High p53 expression was also found to be an independent prognostic factor (p = 0.023). Based on previous data and results provided by this study, p53 expression exerts an independent negative prognostic role in localized prostate cancer and could therefore be evaluated as a useful new molecular marker to be added in the set of known prognostic indicators of the disease. With respect to COX-2 and cyclin D1, further studies are required to elucidate their role in early prediction of PCa relapse after RP.Pathology & Oncology Research 07/2011; 18(2):245-52. · 1.56 Impact Factor
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ABSTRACT: Prostate cancer remains a major health problem in the United States. Established clinicopathologic parameters such as Gleason score, T stage, and prostate-specific antigen levels are currently the guiding tools for prognostication and disease management. The addition of biomarkers could increase the accuracy of these parameters for predicting disease progression, response to therapy, and survival. In this regard, the goal of this study was to evaluate minichromosome maintenance complex protein 2 and Ki-67 immunohistochemical expression as predictors of outcome in prostate cancer. For this purpose, 11 tissue microarrays were constructed using tumor and nontumor samples from 428 patients. Patients were divided into short-term (mean, 2.9 years) and long-term (mean, 14.1 years) follow-up groups. End points were biochemical recurrence for the short-term follow-up group and prostate cancer-related death for the long-term follow-up group. All men in the long-term follow-up group had biochemical recurrence at the time of recruitment. Expression of both markers was higher in tumor than in nontumor glands. Percentage of minichromosome maintenance complex protein 2 was associated with Gleason score in both groups. Percentage of Ki-67 was associated with Gleason score and pathologic stage only in the short-term follow-up group. Higher minichromosome maintenance complex protein 2 percentages were associated with biochemical recurrence in the short-term follow-up group. In the long-term follow-up group, neither minichromosome maintenance complex protein 2 nor Ki-67 levels predicted prostate cancer death. In conclusion, our results suggest that in patients treated by radical prostatectomy for clinically localized prostate cancer, immunohistochemistry for minichromosome maintenance complex protein 2 expression could be used to predict biochemical recurrence, independent of other known clinicopathologic factors.Human pathology 05/2012; 43(11):1852-65. · 3.03 Impact Factor
Abstract. Objective: The study was designed to analyse the
prognostic value of proliferation markers Ki-67 and cyclin D1
and apoptosis in prostate cancer (PC) patients treated by
radical prostatectomy. Patients and Methods: Two hundred
and eleven patients treated by radical prostatectomy for
localised prostate cancer were clinically followed up for a
mean of 7.3 years. The primary histopathological specimens
were re-analysed to ensure uniform histopathological grading
and pT classification. A tissue microarray construction
(TMA) was used in immunohistochemisty to assess the
expression of Ki-67, cyclin D1 and the apoptosis marker Tag.
The results were analysed with light microscopy and the
findings were compared to standard histology, pT and clinical
follow-up data. Results: The co-expression of Ki-67 and cyclin
D1 (p=0.05) was common. High fraction of Ki-67 positive
cells and a high fraction of apoptotic cells were often present
in same tumours (p=0.05). High apoptotic rate was related to
positive surgical margin status (p=0.047). Low expression of
Ki-67 was related to a low Gleason score (p<0.001), an
absence of either capsule penetration (p=0.029) or perineural
invasion (p=0.004). High expression of cyclin D1 was related
to perineural growth (p=0.039). Prostate specific antigen
(PSA) recurrence-free survival (RFS) was predicted by
Gleason grade (p<0.001) and capsule invasion (p=0.006).
High expression of Ki-67 (p=0.03), as well as high apoptotic
rate (p=0.04) were related to a high risk of cancer death. In
multivariate analysis the seminal vesicle invasion was the only
independent predictor of cancer death (p=0.01). Conclusion:
The expression of Ki-67, cyclin D1 and a high apoptotic rate
are related to a malignant phenotype in prostate cancer, but
their prognostic value is inferior to standard histological
Prostate cancer (PC) is the most common malignancy in
men in most civilised countries. It is diagnosed more often
at its early stages and in younger men (1). However, 30% of
the cases are presented with capsule penetration at the time
of curative therapy despite good prognostic signs pre-
operatively (2). In addition 30% of patients treated by
radical prostatectomy develop a biochemical relapse. Pre-
operative prostate specific antigen (PSA), Gleason score
and pT classification are the basis on which the high-risk
patients are identified (2). The clinical significance of rising
PSA after radical prostatectomy has not been clearly
established, since the PSA rise for some men means a lethal
disease with metastases, whilst for others never having any
symptoms or clinical evidence of the disease. A high
Gleason grade and short time to first detectable PSA level
are also used as prognostic factors for a high-risk for
metastatic disease (3). The prognosis of patients with
clinically local tumours cannot be predicted accurately and
more precise methods are needed. Proliferation markers are
good prognostic markers in advanced PC, but their role in
local PC is not well-established in a clinical context.
The proliferation rate can be measured by quantifying
the fraction of Ki-67 antigen-positive cells in immuno-
histochemically prepared specimens. Ki-67 is a molecule
expressed throughout all the points of the cell cycle except
in the G0 resting phase. At early stages of PC
development the proliferation rate is usually low (4).
Proliferation is related to other prognostic factors and to
outcome in PC (4-8), though the number of studies on
local PC is limited (6, 7, 9).
Correspondence to: Sirpa Aaltomaa, MD, Ph.D., Department of
Urology, Kuopio University Hospital, P.O.Box 1777, FIN-70210
Kuopio, Finland. Tel: +358-17-172250, Fax: +358-17-172611,
Key Words: Prostate cancer, radical prostatectomy, prognosis,
Ki-67, apoptosis, cyclin D1.
ANTICANCER RESEARCH 26: 4873-4878 (2006)
Expression of Ki-67, Cyclin D1 and Apoptosis Markers
Correlated with Survival in Prostate Cancer Patients
Treated by Radical Prostatectomy
S. AALTOMAA1, V. KÄRJÄ2, P. LIPPONEN3, T. ISOTALO4,
J-P. KANKKUNEN2, M. TALJA4and R. MOKKA4
Departments of 1Urology and 2Pathology, Kuopio University Hospital, Kuopio;
3Department of Pathology and Forensic Medicin, University of Kuopio, Kuopio;
4Department of Surgery, Päijät-Häme Central Hospital, Lahti, Finland
Cyclin D1 is an amino acid expressed in the G1 phase of
the cell cycle which has an important role in regulating the
cell cycle and cancer progression (10, 11). In
immunohistochemical studies, the overexpression of Cyclin
D1 has been related to cancer progression and outcome in
mixed cohorts of PC patients (10, 11), but the role of cyclin
D1 expression in local PC is unclear.
Apoptosis, or programmed cell death, is essential in the
elimination of damaged cells from the tissues and it
prevents them from replicating. Apoptosis increases parallel
with cell proliferation. If the cell death or proliferation
mechanisms become disturbed for some reason, the tumour
growth can increase and invasion to local structures occurs.
The apoptotic rate, as measured by various methods, is
related to several other prognostic parameters and to
prognosis in PC (5, 8), but its role in early PC is unclear.
The prognostic value of Ki-67, cyclin D1 and apoptosis
was studied in localised PC cases in a cohort treated by
Patients and Methods
Patients. Two hundred and eleven (211) consecutive PC patients
were treated by radical prostatectomy in Kuopio University
Hospital, Finland, between 1987 and 1999, and in Päijät-Häme
Central Hospital, Finland, between 1993 and 1999. The mean (SD)
age of the patients was 64.2 (5.5) years and the mean follow-up was
7.3 (2.4) years. All the patients had a clinically local tumour
according to clinical TNM classification (12). The presence of
distant metastases was excluded by bone scans and chest X-ray
examinations. Adequate histopathological samples for immuno-
histochemistry were available in 184 cases, of which three patients
had lymph node metastases on final histological analysis and were,
therefore, excluded. Seven percent of patients had adjuvant
hormone or radiation therapy.
The follow-up reviews were done at 3-month intervals during the
first year, at 6-month intervals during the next year and annually
thereafter. Recurrent cancers were screened using laboratory tests
(PSA, alkaline phosphatase), digital rectal examination and using
different image analysis methods when required.
Histological methods. Radical prostatectomy specimens were used
for histological analyses. The specimens were fixed in buffered
formalin (pH 7.0), embedded in paraffin, sectioned at 5 Ìm and
stained with haematoxylin and eosin (HE). The specimens were
analysed by two pathologists unaware of the clinical data (VK, J-
PK). The pT classification was done according the UICC 1997
guidelines UICC (12). Histological grading was done according to
Gleason score (13). Capsule invasion, surgical resection margin
status, seminal vesicle invasion and perineural infiltration were
recorded as absent (0) or present (1).
Tissue microarray (TMA) construction. Three representative tumour
regions of each case were marked on HE stained sections. From
these regions, tissue cylinders with diameter of 0.6 mm were
obtained and arrayed into a recipient block using a tissue chip
microarrayer (Beecher Instruments, Silver Spring, MD, USA). The
recipient block was subsequently cut into 5-Ìm sections on
pretreated slides to support adhesion of the tissue samples.
Ki-67 immunohistochemistry. The tissue section was incubated
with monoclonal anti-Ki-67 protein (MIB1, Dianova GmbH,
Germany) antibody diluted at 1:100 in phosphate-buffered saline
(PBS). Several dilutions of the antibody were tested to avoid
background staining and to find optimal staining before the
entire series was processed. Sections were washed twice for 5 min
with PBS, incubated for 20 min with biotinylated secondary
antibody (Vector, CA, USA) diluted in 1:200 in PBS. Slides were
washed twice in PBS for 10 min and incubated for 20 min in a
preformed avidin-biotinylated peroxidase complex (ABC,
Vectastain Elite kit, Vector). Sections were washed twice for 5
min with PBS, developed with diaminobenzidine tetrahydro-
chloride substrate (Sigma, UK), slightly counterstained with
Mayer’s hematoxylin, dehydrated, cleared and mounted. Normal
human tonsil tissue was used as a positive control. The mean
fraction of positive nuclei was estimated, and when at least one
positive nuclei was present, it was estimated at 1%. For the
analysis, the Ki-67 was grouped into two categories 0-5% and
>5%, based on previous studies.
Cyclin D1. Paraffin wax-embedded sections from TMA blocks
were washed twice in PBS and heated in a microwave oven at
600W for three cycles of 5 min each in 0.001 M EDTA (pH 8.0)
for cyclin D1 (NCL-CYCD1-GM, Novocastra, Newcastle-upon-
Tyne, UK). Endogenous peroxidase activity was blocked with 5%
H2O2. After treatment with 1.5% normal horse serum (Zymed,
Histostain-plus bulk kit, Zymed Laboratories Inc., San Francisco,
CA, USA) anti-cyclin D1 monoclonal antibody was applied to
the sections at a dilution of 1:10 in PBS with 1% bovine serum
albumin and incubated for 24 h at 4ÆC. Then, the sections were
washed and biotinylated secondary antibody and avidin-biotin
peroxidase reagent (Zymed) were applied to detect bound
primary antibody. Diaminobenzidine tetrahydrochloride (DAB,
Sigma, St. Louis, MO, USA) was used to demonstrate peroxidase
activity. The slides were counterstained with Mayer’s
haematoxylin, dehydrated, cleared and mounted with DePex
(BDH, Poole, Dorset, UK). Samples from the same series
without primary antibody served as negative controls. Cyclin D1
positive nuclei were counted from all three sections of each case.
The mean fraction of positive nuclei was estimated and, for
statistical analysis, the cyclin D1 was grouped into two
categories: 0-70% and >70%. The initial grouping was into
tertiles, but the two lowest tertiles did not differ in any respect
and, hence, were combined.
Apoptosis detection. The staining procedures were based on the
manufacturer’s instructions (Apoptag, In situ Apoptosis Detection
Kit, Intergen, Oxford, UK). Briefly, after deparaffinisation and
dewaxing in xylene sections were treated with proteinase K (20
Ìg/ml) for 5 min at room temperature, washed twice in 3% hydrogen
peroxide for a further 5 min, and then washed in PBS. The kit was
then applied and the slides immersed in the TdT solution supplied
for 1 h at 37ÆC. The slides were then developed using DAB (Sigma)
and counterstained with Methyl Green, dehydrated, cleared and
mounted with DePex (BDH). Each experiment included a positive
control for apoptosis (intestine) and a negative control (omission of
terminal transferase). The apoptosis index (AI) was calculated as the
ratio of positively stained tumour cells and bodies to all tumour cells
ANTICANCER RESEARCH 26: 4873-4878 (2006)
from three biopsies of each case. The cells were counted under x400
magnification. The mean fraction of positive nuclei was estimated
and when at least one positive nucleus was present, it was estimated
at to 1%. Morphological characteristics of apoptosis were chromatin
condensation, nuclear disintegration and formation of crescent caps
of condensed chromatin at the nuclear periphery. For the statistical
analysis, the apoptotic cells were grouped into two categories: 0-5%
and >5%. The initial grouping was into tertiles but the two highest
tertiles did not differ from each other and, hence, were combined.
Statistical analysis. For statistical analysis the SPSS-X program
package was used. The Chi-square-test was used to analyse the
relationship between the groups. Univariate PSA recurrence-free
survival (RFS) analysis (log-rank analysis) was based on the
Kaplan-Meier method. Multi-variate survival analyses were carried
out according to Cox’s methods. In RFS analysis, only patients with
PSA of zero at the first post-operative analysis were included. A
PSA elevation of 0.2 Ìg/ml or more during the follow-up was
considered as a PSA failure event.
The clinical data of the patients are presented in Table I.
The results of immunohistochemistry are shown in Table II.
The mean (median; range) fraction of cells positively-
stained for Ki-67, cyclin D1 and apoptosis were 4.5% (2.0%;
0-40%), 54.8% (60.0%; 0-95%) and 2.1% (2.0%; 0-10%),
respectively. The expression of Ki-67 and cyclin D1
(p=0.05), as well as Ki-67 and apoptosis (p=0.05) were
correlated to each other, but cyclin D1 and apoptosis were
A high fraction of apoptotic cells in tumours was related
to a positive surgical margin status (p=0.047). A low
expression of Ki-67 was related to a low Gleason score
(p<0.0001), absence of either capsule invasion (p=0.029) or
perineural invasion (PNI) (p=0.004). On the other hand,
high expression of cyclin D1 was related to PNI (p=0.039).
PSA recurrence was not predicted by cyclin D1, Ki-67
expression or the fraction of apoptotic cells, whereas the
Gleason score (p=0.03) and capsule invasion (p=0.01) were
significant predictors of PSA recurrence.
Short PSA recurrence free survival was predicted by a
high Gleason grade p<0.0001 and capsule invasion
(p=0.01). The Gleason score (p=0.05), perineural invasion
(p=0.03), capsule invasion (p=0.006), positive surgical
margin status (p=0.01) and seminal vesicle invasion
(p<0.001) were all significant predictors of cancer specific
survival (Table III).
PC related survival time was predicted by Ki-67
expression. The mean PC survival time was 15.6 years in
patients with a low Ki-67 expression as compared to 10.7
years among patients with a high Ki-67 expression (p=0.03,
Figure 1). No PC deaths occurred in patients with low
apoptosis rates as compared to a PC survival time of 14.1
years among men whose tumours showed a high apoptosis
rate (p=0.04, Figure 2).
Cox’s multivariate survival analysis was carried out
including the following prognostic parameters: pT-category,
pre-operative PSA value, Gleason score, capsule invasion,
surgical margin status, seminal vesicle invasion and PNI,
Aaltomaa et al: Proliferation and Apoptosis in Local Prostate Cancer
Table I. The clinical charasteristics of the patients.
Mean age, years (SD)
Mean follow-up, years (SD)
Mean PSA at diagnosis, Ìg/l (SD)
PSA Ìg/ml, n (%)
pT category, n (%)
Gleason score, n (%)
Capsule invasion, n (%)*
Surgical margin status, n (%) *
Seminal vesicle involvement, n (%) *
Postoperative PSA < 0.1 Ìg/l
Survival, n (%)
*data not available in all cases.
Table II. The results of Ki-67, apoptosis and Cyclin D1 immuno-
(% positively stained)
Ki-67, mean 2%
Apoptosis, mean 2%
Cyclin D1, mean 55%
Cyclin D1, Ki-67 and apoptosis rate. The only independent
predictor of PC survival was seminal vesicle invasion
(‚=–2.830, SE 1.096, p=0.010, exp (‚) =0.059; 95%
confidence interval for Exp (‚) =0.007-0.506).
About 30% of radically treated patients will have a
biochemical relapse, which is the first sign of threatening
local or systemic relapse (2). Due to the earlier diagnosis
and younger age of PC patients, the criteria of adjuvant
therapy are under debate. The most important pre-
operatively available prognostic parameter is the Gleason
score (13), but the Gleason score in biopsy specimens is
under- or over-graded in about 30% of cases as compared
to radical prostatectomy specimen analysis. Therefore, new
accurate prognostic markers are urgently needed.
The proliferative activity of PC is on low average
compared to some other tumours and local PCs are
particularly slow proliferators (4, 8, 14, 15). Although this
series included only 40% of patients with a pre-operative
PSA value under 10, the mean fraction of proliferating
cells was only 2%, which is in line with the previous
reports (14). The expression of cyclin D1 and Ki-67 were
ANTICANCER RESEARCH 26: 4873-4878 (2006)
Table III. The significant parameters related to prostate cancer specific
Mean time (SE)95% CI
Seminal vesicle invasion
no PC deaths
no PC deaths
14.1 (0.54) 13.1-15.2
no PC deaths
PC: prostate cancer.
Figure 1. The cancer related survival categorised according to the fraction
of Ki-67 positive cells (p=0.03). Ki-67 0-5%, n=156; Ki-67> 5%, n=25.
Figure 2. The cancer-related survival categorised according to the fraction
of apoptotic cells (p=0.04). Apoptosis rate 0-5%, n=65; apoptosis rate >
significantly interrelated, which confirms the findings by
Drobnjak et al.
(10). There was a significant
interrelationship between high Ki-67 and strong apoptotic
activity, which is also in line with previous reports (5).
Vesalainen et al. found a correlation between a high
apoptosis rate and a high Gleason score in a series
including all stages of PC (16), whereas in our series
including mainly local PCs, Gleason score and apoptosis
were not significantly interrelated. Apoptotic index seems
to be different (17) in local and disseminated cancers,
which most probably explains the different results.
Considerable variation also exists, depending on the
method of apoptosis detection used.
The higher the proliferation rate, the higher the
proportion of cells referring to apoptotic cell death (5).
Both Ki-67 and the proportion of apoptotic cells predicted
PC specific survival. No deaths from PC occurred in the
group of patients with low apoptotic activity and deaths
were also rare in the group patients of tumours with a low
proliferation rate. Similar survival data in mixed cohorts and
even independent prognostic value for Ki-67 expression (4,
7, 9, 14) and apoptosis (18) have been reported in literature
In a mixed cohort, including a large number of PC
patients, the AI based on morphological criteria alone had
prognostic value (16). The higher the AI, the higher was the
probability of cancer death. Pollack et al. found prognostic
value for Ki-67 using the cut-off value of 7.1% of
proliferating cells. Ki-67 expression predicted biochemical
failure, distant metastasis, and cancer-specific death in
patients treated by radiotherapy (14). All these results are
in line with the current survival data.
The Gleason grade is a well-established prognostic factor
in PC, whereas the role of PNI is questioned in the
literature (16). We found a positive correlation between Ki-
67, pT and differentiation of tumours, which has been
established previously (4). A positive correlation with PNI
and cyclin D1 was also found. So it would seem that a high
expression of Ki-67 and cyclin D1 are related to other
malignant histological features. The co-expression of cyclin
D1 and Ki-67 has been reported previously (10) and
confirms our current results. In contrast to disseminated
cancers (11), in local tumours cyclin D1 expression seems to
have no prognostic value.
This study is based on TMA construction. The tissue
block in final analysis is small compared to that used in
standard immunohistochemistry, where the entire tissue
section is available for analysis. However, in TMA the
staining results are more standardised, which is an
advantage although information may be missed due to
small amounts of tissue. These methodological facts must
be taken into account when comparing current results to
Ki-67, cyclin D and apoptotic markers are statistically
significant prognostic factors in an univariate analysis, but
in multivariate analysis only seminal vesicle invasion
remains statistically significant.
This study was financially supported by a research grant (EVO
funding) from the Kuopio University Hospital and Päijät Häme
Central Hospital. The technical assistance of Assistant Professor
Irina Alafuzoff and Mrs Helena Kemiläinen are gratefully
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Received May 29, 2006
Revised September 13, 2006
Accepted September 18, 2006
ANTICANCER RESEARCH 26: 4873-4878 (2006)