Yoshimi Ozaki-Ohgami et al.

when > 50% of tumor cells are stained. Thestaining pattern was classified as follows: whenmore than ≥ 50% of the tumor cells showed positive staining, labeling was considered positive,and when < 50% of the tumor cells showed positive staining, labeling was considered negative.

Target Retrieval Solution (DAKO, Carpentaria,CA) for retrieval of antigens. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide
in methanol for 20 min. Tissue sections werewashed three times with phosphate-buffered saline (PBS) and pre-blocked for 60 min with 10%
goat serum. After washing with PBS, the samples
were incubated at 4°C for 48 h with the following primary antibodies: polyclonal antibodies
rose against p-Akt (Ser 473) (diluted 1:100; Cell
Signaling Technology, Beverly, MA) and PTEN
(diluted 1:100; Upstate Cell Signaling Solutions,Charlottesville, VA). Sections were washed threetimes in PBS and incubated with the secondary
antibody, anti-rabbit immunoglobulin (IgG + IgA+ IgM) conjugated with biotin (Nichirei) for 60min, followed by incubation with a streptavidinperoxidase complex (Nichirei) for 60 min. After
three additional washes in PBS, diaminobenzidine
tetrahydrochloride working solution was applied.
Finally, the slides were counterstained with hematoxylin.
All immunostained sections were evaluatedin a blinded manner with no knowledge of clinical and pathological factors. p-Akt and PTEN
expression was not detected in the non-neoplastic
region of the pancreas. The intensity of staining
seen in different areas of the same slide was analyzed using previously reported criteria (Malik etal., 2002). The intensity is designated as negative
when no tumor cells are stained, weak when 10–20% of tumor cells are stained, moderate when
20–50% of tumor cells are stained, and strong

Statistical analysis

Statistical analysis included the standard chisquare test and Mann-Whitney U test. The survival rates were estimated by the Kaplan-Meiermethod, and statistical analysis was carried out
by the log-rank test for equality of the survival
curves. In multivariate analysis, the influence of each variable on survival was assessedby the Cox proportional hazards model and astepwise procedure (StatView 5.0; Abacus Concepts Berkeley, CA). Statistical significancewas considered with P < 0.05.
Results
Immunohistochemistry showed that 32 (62.7%)of the 51 tumors demonstrated p-Akt-positiveexpression, and 26 (51%) demonstrated PTENpositive expression (Table 1). The typical patternof p-Akt and PTEN staining included both cytoplasic and nuclear immunostaining (Fig. 1).
A tendency towards some close correlation
between p-Akt and PTEN expression was noticeable, but there was no signiﬁcant relationship between p-Akt and PTEN expression (P = 0.0549;
Table 1). The correlation between p-Akt orPTEN expression and clinicopathological factors
is shown in Table 2. p-Akt expression showed nocorrelations with age, gender, histology, venous
invasion or lymph node metastasis, whereas it wassigniﬁcantly associated with tumor size, arterial
invasion and pathological stage. These data suggest that p-Akt expression was correlated with tumor progression in pancreatic cancer. On the other hand, PTEN expression showed no correlation
with age, tumor size, gender, histology, venous

Table 1. Correlations between p-Akt and PTEN
expressions
PTEN
Negative
Positive
p-Akt-negative
p-Akt-positiveTotal

61925 (49%)

131326 (51%)

Total
19 ( 37.3%)
32 ( 62.7%)
51 (100%)

There was a tendency towards a close correlation between p-Akt and PTEN expression, but the relationshipbetween p-Akt and PTEN expression was not signiﬁcant(P = 0.0549).

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p-Akt and PTEN expression in pancreatic cancer

Table 2. Correration between expressions of p-Akt or PTEN and clinicopathological factors in 51
patients with pancreatic cancer
Variablep-Akt
Positive (%) Negative (%) P valueAge† (year)< 65
11 (34.4)≥ 65
21 (67.4)
Tumor size‡ (cm)< 4.0
13 (44.8)≥ 4.0
16 (55.2)
GenderMale
25 (78.1)
Female7 (21.9)
Histology: Invasive ductal carcinomaGood
10 (31.3)
Moderate
20 (62.5)
Poor2 ( 6.3)
Arterial invasion
Positive
15 (46.9)
Negative
17 (53.1)
Venous invasion
Positive
14 (43.8)
Negative
18 (56.3)
Lymph node metastasis
Positive
23 (71.9)
Negative
9 (28.1)
Stage (pTNM)
I
1 ( 3.1)II
4 (12.5)III
5 (15.6)
IV22 (68.8)

PTEN
Positive (%) Negative (%) P value

9 (47.4)
10 (52.6)NS

11 (42.3)
15 (57.7)

9 (36.0)
16 (64.9)NS

14 (73.7)
5 (26.3)

0.048714 (56.0)
11 (44.0)

13 (56.5)
10 (43.5)

NS

13 (64.8)
6 (31.6)

NS

18 (69.2)
8 (30.8)

20 (80.0)
5 (20.0)NS

7 (36.8)9 (47.4)
3 (15.8)NS

8 (30.8)
14 (53.8)
4 (15.4)

9 (36.0)
15 (60.0)
1 ( 4.0)NS

3 (15.8)
16 (84.2)

0.02475 (19.2)
21 (80.8)

13 (52.0)
12 (48.0)

0.01445 (26.3)
14 (73.7)NS

7 (26.9)
19 (73.1)

12 (48.0)
13 (52.0)NS

14 (73.7)
5 (26.3)

NS

16 (61.5)
10 (38.5)

21 (84.0)
4 (16.0)NS

0.0233

1 ( 3.8)
1 ( 3.8)
11 (42.3)
13 (50.0)

0 ( 0.0)
3 (12.0)
5 (20.0)
17 (68.0)

NS0 ( 0.0)0 ( 0.0)
11 (57.9)
8 (42.1)† Mean ± SD, 65.2 ± 8.0 years.
‡ Mean ± SD, 3.9 ± 1.3 cm.
NS, not signiﬁcant; pTNM, pathological tumor-node-metastasis.
p-Akt-negative expression was more frequently detected in smaller size tumors and in none of the cases with arterial invasion. p-Akt-positive expression was more frequently detected in advanced histological stage tumors. PTENpositive expression was detected signiﬁcantly more frequently in patients with no arterial invasion.

invasion, lymph node metastasis or pathologicalstage, whereas it was signiﬁcantly associated witharterial invasion (P = 0.0144).
Postoperative survival curves for patientswith pancreatic cancer according to p-Akt andPTEN expression are shown in Figs. 2 and 3, respectively. The estimated 5-year survival rate forpatients with negative or positive p-Akt expression
was 25.7 and 11.3%, respectively. Patients with
p-Akt-positive tumors had significantly poorer
prognoses than those with p-Akt-negative tumors(P = 0.0132; Fig. 2). The estimated 5-year survival rate for patients with negative and positive
PTEN expression was 0 and 29.9%, respectively.

Fig. 2. Post-operative survival curves of patients withpancreatic cancer according to expression of p-Akt. Patients with positive p-Akt tumors had signiﬁcantly worse
prognoses than those with p-Akt-negative tumors (P =0.0132). [ ], number of cases.

13