Int J Clin Exp Pathol 2013;6(10):2112-2120 www.ijcep.com /ISSN:1936-2625/IJCEP1308017
Original Article Clinicopathological significance of PTEN and PI3K/AKT signal transduction pathway in non-small cell lung cancer Fen Yun1, Yongfeng Jia1, Xiuxia Li1, Li Yuan2, Qinnuan Sun1, Huiling Yu1, Lin Shi1, Hongwei Yuan1 Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical University, Huhhot, 010059, China; 2Shandong Heze Medical College, Heze, Shandong 274000 1
Received August 8, 2013; Accepted September 3, 2013; Epub September 15, 2013; Published October 1, 2013 Abstract: A high frequency of mutations at the PTEN locus has been noticed in carcinoma of lung. However, the role of PTEN alternations and its association with outcome variables in the genesis of lung carcinoma are not understood fully. The purpose of our study was to examine the impact of EGFR, TGF-α, P-AKT and PTEN in the genesis of non-small cell lung cancer (NSCLC). Total numbers of 66 histopathologically confirmed cases of NSCLC and 10 cases of benign control samples embedded with wax were studied. We assessed EGFR, TGF-α and P-AKT by the use of specific antibody through immunohistochemistry as directed by the manufacturer, and detected PTEN expression by in situ hybridization. There were progressive loss of PTEN expression and significant increasing in EGFR, TGF-α, P-AKT expression from benign samples to NSCLC (p53
32
17
Male
32
18
Female
12
4
X2
P-value*
0.158
0.691
0.66
0.417
5.961
0.015
4.714
0.03
8.727
0.003
Age
Sex
Lymph node metastasis Positive
28
7
Negative
16
15
Pathological pattern adenocarcinoma
20
4
Squamous carcinoma
24
18
Moderate-well
24
20
Poor
20
2
Differentiation grade
Note: *Chi-square test.
2113
This study attempts to study the differential expression pattern of EGFR, TGF-α and P-AKT and PTEN protein for its relevance in development and progression of NSCLC. Materials and methods Study population A total of 66 (50 males and 16 females) of histopathologically confirmed cases of NSCLC, and 10 cases of benign control samples of lung were assessed for EGFR, TGF-α, P-AKT and PTEN expression. The age of the patients ranged from 28-77 years with a mean age of 53 years, The patients were diagnosed squamous cell carcinoma (n=42) and adenocarcinoma (n=24), and 41 in advanced stages III/IV, with 25 patients in stages I/II, according to TNM classification. Histopathologic-
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer
Figure 2. EGFR expression in NSCLC and benign samples of lung (A: Negative expression of EGFR in lung normal tissue; B: Positive expression of EGFR in Squamous carcinoma tissue; C: Positive expression of EGFR in lung adenocarcinoma tissue. Original magnification ×400).
Table 3. EGFR expression in NSCLC and benign samples Tissue
EGFR
X
P-value
4.367
0.037
2
+
-
Positive ratio (%)
NSCLC
46
20
69.70%
Benign sample
3
7
30.00%
*
Note: *Chi-square test.
Table 4. Correlation between EGFR and clinicopathological factors Factors
EGFR -
+~+++
X2
P-value*
0.27
0.603
0.047
0.828
12.569
0.001
0.164
0.686
7.03
0.008
Age ≤53
6
11
>53
14
35
Male
16
34
Female
4
12
Sex
Lymph node metastasis Positive
4
31
Negative
16
15
Pathological pattern adenocarcinoma
8
16
Squamous carcinoma
12
30
Moderate-well
18
26
Poor
2
20
Differentiation grade
Note: *Chi-square test.
2114
ally, the NSCLCs were categorized as well differentiated and moderately differentiated -44 cases and poorly differentiated -22 cases, 35 cases with and 31 cases without lymph node metastasis. The benign samples of lung (males and females), 31-65 years of age (median age, 48 years) were taken as control. The protocol of this study was approved by the Protocol Review Committee and the Bioethics Committee of Inner Mongolia Medical University. PTEN in situ hybridization In order to detect PTEN expression, in situ hybridization assays were per-
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer
Figure 3. TGF-α expression in NSCLC and benign samples of lung (A: Negative expression of TGF-α in lung normal tissue; B: Positive expression of TGF-α in lung Squamous carcinoma tissue; C: Positive expression of TGF-α in lung adenocarcinoma tissue. Original magnification ×400).
Table 5. TGF-α expression in cytoplasm of NSCLC and benign samples Tissue
TGF-α +
-
NSCLC
47
19
71.20%
Benign sample
3
7
30.00%
X2
P-value*
4.850
0.028
Positive ratio (%)
Note: *Chi-square test.
Table 6. Correlation between TGF-α and clinicopathological factors Factors
TGF-α -
+~+++
X2
P-value*
0.142
0.706
0
1
7.645
0.006
1.396
0.237
3.695
0.055
Age ≤53
6
11
>53
13
36
Male
14
36
Female
5
11
Positive
5
30
Negative
14
17
adenocarcinoma
9
15
Squamous carcinoma
10
32
Moderate-well
16
28
Poor
3
19
Sex
Lymph node metastasis
Pathological pattern
Differentiation grade
Note: *Chi-square test.
2115
formed on serial 5 microns thick sections from the original blocks and the TMA block, using a kit for in situ hybridization (MK1276, Boster Biotech Co, Wuhan, China), according to the provider’s specifications as described in detail elsewhere. The poly-A probe, used as an indicator of the preservation of mRNA in the cells (positive control), resulted mainly in dark brown nuclear staining and less in a cytoplasmic one. Immunohistochemical analysis Formalin fixed paraffinembedded tissue blocks were cut in 5 microns thick serial sections. The sections were deparaffinized, rehydrated and rinsed in
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer
Figure 4. P-AKT immunostaining showing expression in cytoplasm of NSCLC and benign samples of lung (A: Negative expression of P-AKT in lung normal tissue; B: Positive expression of P-AKT in lung Squamous carcinoma tissue; C: Positive expression of P-AKT in lung adenocarcinoma tissue. Original magnification ×400).
Table 7. P-AKT expression in cytoplasm of NSCLC and benign samples Tissue
P-AKT +
-
Positive ratio (%)
NSCLC
50
16
75.80%
Benign sample
2
8
20.00%
X2
P-value*
10.048
0.002
Note: *Chi-square test.
Table 8. Correlation between P-AKT and clinicopathological factors Factors
P-AKT -
+~+++
X2
P-value*
0.166
0.683
0
1
6.662
0.010
2.832
0.092
4.125
0.042
Age ≤53
3
14
>53
13
36
Male
12
38
Female
4
12
Sex
Lymph node metastasis Positive
4
31
Negative
12
19
Pathological pattern adenocarcinoma
3
21
Squamous carcinoma
13
29
Moderate-well
14
30
Poor
2
20
Differentiation grade
Note: *Chi-square test.
2116
phosphate buffer saline (PBS). An Immunohistochemical assay for EGFR, TGFα, P-AKT was performed on consecutive paraffin sections using streptavidin-biotin method. Monoclonal mouse antihuman EGFR antibody (ZM0093, Beijing ZSGB Company, China), monoclonal rabbit antihuman TGF-α antibody (ZA254, Beijing ZSGB Company, China) and monoclonal mouse antihuman P-AKT antibody (sc16646, Beijing ZSGB Company, China) were used as primary antibodies respectively. After antigen retrieval slides were incubated with primary antibody, followed by secondary biotinylated antibody. Sections were washed in PBS and then incubated
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer Scoring method
Table 9. Correlation between EGFR and TGF-α expression EGFR + -
TGF-α +
-
37 10
9 10
X2
P-value*
0.309
0.012
Note: *Chi-square test.
Table 10. Correlation between EGFR and P-AKT expression EGFR + -
P-AKT + 39 11
7 9
X2
P-value*
0.319
0.022
Note: *Chi-square test.
Table 11. Correlation between P-AKT and TGF-α expression P-AKT + -
TGF-α + 41 6
9 10
X2
P-value*
0.421
0.002
Statistical analysis
Table 12. Correlation between EGFR and PTEN expression
+ -
PTEN + 9 13
37 7
X2
P-value*
-0.443
0.001
Note: *Chi-square test.
Table 13. Correlation between TGF-α and PTEN expression TGF-α + -
PTEN + 11 11
36 8
X2
P-value*
-0.331
0.007
Loss of PTEN expression in NSCLC
Table 14. Correlation between P-AKT and PTEN expression
+ -
PTEN + 12 10
38 6
X2
P-value*
-0.35
0.004
Note: *Chi-square test.
with streptavidin peroxidase. Finally chromogen Diaminobenzidine (DAB) was used and section were counterstain with hematoxylin.
2117
Chi-square (X)2 test was performed to find out the possible correlation among EGFR, TGF-α, P-AKT and PTEN and other clinical parameters in NSCLC and benign samples of lung tissue. We used the SPSS ver. 14.0 (SPSS Inc., Chicago, IL, USA), with P
Original Article Clinicopathological significance of PTEN and PI3K/AKT signal transduction pathway in non-small cell lung cancer Fen Yun1, Yongfeng Jia1, Xiuxia Li1, Li Yuan2, Qinnuan Sun1, Huiling Yu1, Lin Shi1, Hongwei Yuan1 Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical University, Huhhot, 010059, China; 2Shandong Heze Medical College, Heze, Shandong 274000 1
Received August 8, 2013; Accepted September 3, 2013; Epub September 15, 2013; Published October 1, 2013 Abstract: A high frequency of mutations at the PTEN locus has been noticed in carcinoma of lung. However, the role of PTEN alternations and its association with outcome variables in the genesis of lung carcinoma are not understood fully. The purpose of our study was to examine the impact of EGFR, TGF-α, P-AKT and PTEN in the genesis of non-small cell lung cancer (NSCLC). Total numbers of 66 histopathologically confirmed cases of NSCLC and 10 cases of benign control samples embedded with wax were studied. We assessed EGFR, TGF-α and P-AKT by the use of specific antibody through immunohistochemistry as directed by the manufacturer, and detected PTEN expression by in situ hybridization. There were progressive loss of PTEN expression and significant increasing in EGFR, TGF-α, P-AKT expression from benign samples to NSCLC (p53
32
17
Male
32
18
Female
12
4
X2
P-value*
0.158
0.691
0.66
0.417
5.961
0.015
4.714
0.03
8.727
0.003
Age
Sex
Lymph node metastasis Positive
28
7
Negative
16
15
Pathological pattern adenocarcinoma
20
4
Squamous carcinoma
24
18
Moderate-well
24
20
Poor
20
2
Differentiation grade
Note: *Chi-square test.
2113
This study attempts to study the differential expression pattern of EGFR, TGF-α and P-AKT and PTEN protein for its relevance in development and progression of NSCLC. Materials and methods Study population A total of 66 (50 males and 16 females) of histopathologically confirmed cases of NSCLC, and 10 cases of benign control samples of lung were assessed for EGFR, TGF-α, P-AKT and PTEN expression. The age of the patients ranged from 28-77 years with a mean age of 53 years, The patients were diagnosed squamous cell carcinoma (n=42) and adenocarcinoma (n=24), and 41 in advanced stages III/IV, with 25 patients in stages I/II, according to TNM classification. Histopathologic-
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer
Figure 2. EGFR expression in NSCLC and benign samples of lung (A: Negative expression of EGFR in lung normal tissue; B: Positive expression of EGFR in Squamous carcinoma tissue; C: Positive expression of EGFR in lung adenocarcinoma tissue. Original magnification ×400).
Table 3. EGFR expression in NSCLC and benign samples Tissue
EGFR
X
P-value
4.367
0.037
2
+
-
Positive ratio (%)
NSCLC
46
20
69.70%
Benign sample
3
7
30.00%
*
Note: *Chi-square test.
Table 4. Correlation between EGFR and clinicopathological factors Factors
EGFR -
+~+++
X2
P-value*
0.27
0.603
0.047
0.828
12.569
0.001
0.164
0.686
7.03
0.008
Age ≤53
6
11
>53
14
35
Male
16
34
Female
4
12
Sex
Lymph node metastasis Positive
4
31
Negative
16
15
Pathological pattern adenocarcinoma
8
16
Squamous carcinoma
12
30
Moderate-well
18
26
Poor
2
20
Differentiation grade
Note: *Chi-square test.
2114
ally, the NSCLCs were categorized as well differentiated and moderately differentiated -44 cases and poorly differentiated -22 cases, 35 cases with and 31 cases without lymph node metastasis. The benign samples of lung (males and females), 31-65 years of age (median age, 48 years) were taken as control. The protocol of this study was approved by the Protocol Review Committee and the Bioethics Committee of Inner Mongolia Medical University. PTEN in situ hybridization In order to detect PTEN expression, in situ hybridization assays were per-
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer
Figure 3. TGF-α expression in NSCLC and benign samples of lung (A: Negative expression of TGF-α in lung normal tissue; B: Positive expression of TGF-α in lung Squamous carcinoma tissue; C: Positive expression of TGF-α in lung adenocarcinoma tissue. Original magnification ×400).
Table 5. TGF-α expression in cytoplasm of NSCLC and benign samples Tissue
TGF-α +
-
NSCLC
47
19
71.20%
Benign sample
3
7
30.00%
X2
P-value*
4.850
0.028
Positive ratio (%)
Note: *Chi-square test.
Table 6. Correlation between TGF-α and clinicopathological factors Factors
TGF-α -
+~+++
X2
P-value*
0.142
0.706
0
1
7.645
0.006
1.396
0.237
3.695
0.055
Age ≤53
6
11
>53
13
36
Male
14
36
Female
5
11
Positive
5
30
Negative
14
17
adenocarcinoma
9
15
Squamous carcinoma
10
32
Moderate-well
16
28
Poor
3
19
Sex
Lymph node metastasis
Pathological pattern
Differentiation grade
Note: *Chi-square test.
2115
formed on serial 5 microns thick sections from the original blocks and the TMA block, using a kit for in situ hybridization (MK1276, Boster Biotech Co, Wuhan, China), according to the provider’s specifications as described in detail elsewhere. The poly-A probe, used as an indicator of the preservation of mRNA in the cells (positive control), resulted mainly in dark brown nuclear staining and less in a cytoplasmic one. Immunohistochemical analysis Formalin fixed paraffinembedded tissue blocks were cut in 5 microns thick serial sections. The sections were deparaffinized, rehydrated and rinsed in
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer
Figure 4. P-AKT immunostaining showing expression in cytoplasm of NSCLC and benign samples of lung (A: Negative expression of P-AKT in lung normal tissue; B: Positive expression of P-AKT in lung Squamous carcinoma tissue; C: Positive expression of P-AKT in lung adenocarcinoma tissue. Original magnification ×400).
Table 7. P-AKT expression in cytoplasm of NSCLC and benign samples Tissue
P-AKT +
-
Positive ratio (%)
NSCLC
50
16
75.80%
Benign sample
2
8
20.00%
X2
P-value*
10.048
0.002
Note: *Chi-square test.
Table 8. Correlation between P-AKT and clinicopathological factors Factors
P-AKT -
+~+++
X2
P-value*
0.166
0.683
0
1
6.662
0.010
2.832
0.092
4.125
0.042
Age ≤53
3
14
>53
13
36
Male
12
38
Female
4
12
Sex
Lymph node metastasis Positive
4
31
Negative
12
19
Pathological pattern adenocarcinoma
3
21
Squamous carcinoma
13
29
Moderate-well
14
30
Poor
2
20
Differentiation grade
Note: *Chi-square test.
2116
phosphate buffer saline (PBS). An Immunohistochemical assay for EGFR, TGFα, P-AKT was performed on consecutive paraffin sections using streptavidin-biotin method. Monoclonal mouse antihuman EGFR antibody (ZM0093, Beijing ZSGB Company, China), monoclonal rabbit antihuman TGF-α antibody (ZA254, Beijing ZSGB Company, China) and monoclonal mouse antihuman P-AKT antibody (sc16646, Beijing ZSGB Company, China) were used as primary antibodies respectively. After antigen retrieval slides were incubated with primary antibody, followed by secondary biotinylated antibody. Sections were washed in PBS and then incubated
Int J Clin Exp Pathol 2013;6(10):2112-2120
PTEN and PI3KAKT pathway in non-small cell lung cancer Scoring method
Table 9. Correlation between EGFR and TGF-α expression EGFR + -
TGF-α +
-
37 10
9 10
X2
P-value*
0.309
0.012
Note: *Chi-square test.
Table 10. Correlation between EGFR and P-AKT expression EGFR + -
P-AKT + 39 11
7 9
X2
P-value*
0.319
0.022
Note: *Chi-square test.
Table 11. Correlation between P-AKT and TGF-α expression P-AKT + -
TGF-α + 41 6
9 10
X2
P-value*
0.421
0.002
Statistical analysis
Table 12. Correlation between EGFR and PTEN expression
+ -
PTEN + 9 13
37 7
X2
P-value*
-0.443
0.001
Note: *Chi-square test.
Table 13. Correlation between TGF-α and PTEN expression TGF-α + -
PTEN + 11 11
36 8
X2
P-value*
-0.331
0.007
Loss of PTEN expression in NSCLC
Table 14. Correlation between P-AKT and PTEN expression
+ -
PTEN + 12 10
38 6
X2
P-value*
-0.35
0.004
Note: *Chi-square test.
with streptavidin peroxidase. Finally chromogen Diaminobenzidine (DAB) was used and section were counterstain with hematoxylin.
2117
Chi-square (X)2 test was performed to find out the possible correlation among EGFR, TGF-α, P-AKT and PTEN and other clinical parameters in NSCLC and benign samples of lung tissue. We used the SPSS ver. 14.0 (SPSS Inc., Chicago, IL, USA), with P
