299
Cancer Biomarkers 13 (2013) 299–305 DOI 10.3233/CBM-130352 IOS Press
Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma detected by quantum dots-based immunofluorescence technology Yi Zhanga,1, Xue Yaoa,1 , Congqing Jiangb , Junqiu Yuec, Jing Guand , Honglei Chenga, Mohammed Hajirashida , Yan Wanga and Lifang Fana,∗ a
Department of Pathology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, China Department of General Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China c Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei, China d Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China b
Abstract. BACKGROUND: Small intestinal adenocarcinoma (SIA) is often diagnosed at an advanced stage due to its rarity and nonspecific clinical manifestations. Abnormal activation of the PI3K/Akt pathway may lead to various diseases including cancer. In this study, we explored the relationship between those pathways and SIA. OBJECTIVE: To investigate the expression of PI3K, phospho-Akt (p-Akt) and PTEN in SIA to evaluate their clinical significance. METHODS: Expression of PI3K, p-Akt and PTEN biomarkers were examined by quantum dots-based immunofluorescence histochemistry (QDs-IHC) and traditional immunohistochemistry (IHC). There are 53 SIA samples, 11 non-cancer tissue samples and 11 normal small intestine tissues. RESULTS: The positive frequencies of PI3K and p-Akt expression were significantly higher in the SIA group (77.4%, 41/53 and 77.4%, 41/53, respectively) than that in the non-cancer group (36.4%, 4/11 and 27.3, 3/11, respectively) and normal small intestine tissue group (36.4%, 4/11 and 18.2, 2/11, respectively). However, the difference of the positive rates of PTEN expression in three groups did not reach a statistical difference (P = 0.761). The IHC results were in concordance with the QDs-IHC. CONCLUSIONS: These observations support that PI3K/Akt pathway is highly activated in SIA, which may implicate therapeutic targeting of the PI3K/Akt signaling pathway for its treatment. Keywords: Adenocarcinoma, PI3K, PTEN, p-Akt, quantum dots, small intestine
1. Introduction Small intestine accounts for 75% of the length of gastrointestinal tract and over 90% of the mucosal sur1 These
authors contributed equally to this study. author: Lifang Fan, Department of Pathology, School of Basic Medical Science, Wuhan University, 185 Donghu Road, Wuchang District, 430071 Wuhan, Hubei, China. E-mail: [email protected]. ∗ Corresponding
face. But only 2%–3% of malignancies of the gastrointestinal system happen in the small bowel, whereas, colorectal carcinoma is the third most common cancer worldwide. It has been confirmed that SIA showed different immunophenotypes compared with colorectal adenocarcinoma, though SIA is morphologically similar to colorectal adenocarcinoma and shares many of the risk factors in tumorigenesis [1]. Little research has been done into the molecular mechanisms of SIA due to the lack of samples.
c 2013 – IOS Press and the authors. All rights reserved ISSN 1574-0153/13/$27.50
300
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma
PI3K/Akt pathway plays an important role in pathogenesis of many human cancers, and is involved in DNA synthesis and cell proliferation, and PTEN is a major negative regulator of the PI3K/Akt signaling pathway. Abnormal activation of the pathway may lead to various diseases including cancer [2]. However, few studies about this pathway in SIA have been carried out. QDs-IHC is a newly developed technique to detect the expressions of proteins. It can stain both superficial and deeper parts of the tumor cells, and the QD fluorescence of the tumor cell remains unchanged when background tissue autofluorescence changes with the lengths of different excitation waves, which makes it much easier to detect even a very small nest of tumor cells [3]. Previous studies demonstrated that QDsIHC possess advantages of a higher fluorescent efficiency, greater adaptability, higher photostability and longer preservation, making it more sensitive and accurate than conventional immunohistochemistry, especially in the evaluation of low-expressed genes [4,5]. SIA is often diagnosed at an advanced stage due to its rarity and nonspecific clinical manifestations, which contributes to a poor prognosis. Hereby, we performed quantum dots-based immunofluorescence in the current study to investigate the expressions of PI3K, p-Akt and PTEN in samples of SIA patients, in an attempt to help with the clinical treatment of SIA. The results turn out that PI3K and p-Akt proteins are overexpressed in SIA samples.
2. Materials and methods 2.1. Tissue samples Small intestine disease spectrum tissue microarrays (TMAs) were obtained from US Biomax Inc. (http://www.biomax.us/tissue-arrays/Intestine/SM802; lot number SM802). Tissue samples were arranged in 10 columns and 8 rows for a total of 80 individual cores (1.5 mm, 5 μm). 5 non-small intestine samples were excluded, and a total of 75 cores of small intestine specimen were examined in this study. 53 patients were grouped into SIA group, in which 45 cases were diagnosed with primary SIA and 8 cases were metastasized to omentum or lymph nodes. 3 cases with ulcer at small intestine and 8 with chronic inflammation were grouped into non-cancer group. 5 samples of normal small intestine tissue and 6 cancer adjacent normal small intestine tissues were grouped into normal tissue group.
2.2. Quantum dots-based immunofluorescence histochemistry (QDs-IHC) QDs-IHC was performed according to the manufacturer’s instructions (Wuhan Jiayuan Quantum Dot Co, LTD, China). The 5 μm TMAs were deparaffinized in xylene and microwaved for 15 minutes in citric acid buffer (10 mM, pH 6.0) for antigen retrieval. For antibody bindings, TMAs were first incubated in 10% normal goat serum for 30 minutes to block nonspecific binding and then at 4◦ C overnight in primary rabbit anti-Phospho-Akt polyclonal antibody (1:50 dilution, #3787, cell signaling, USA), rabbit anti-PTEN polyclonal antibody (1:200 dilution, #9559, cell signaling, USA) and rabbit anti-PI3 Kinase p110α polyclonal antibody (1:400 dilution, #4249, cell signaling, USA), respectively. TMAs were then washed with TBS (0.5% Tween, 0.1 MTris-base, 0.9% NaCl, and pH 7.6) and incubated in biotinylated goat anti-rabbit IgG (1:100 dilution, Jackson Immuno Research, West Grove, A). Control groups were prepared in parallel but incubated in TBS instead of primary antibody. The TMAs were again washed in TBS and finally incubated with the QDs conjugated streptavidin (QDs-SA) probes (605nm-QDs-SA, cat. no. L-1002-1), prepared by Wuhan Jiayuan Quantum Dots Co., Ltd (Wuhan, China) for 30 min at 37◦ C. After washing in TBS and sealing with 90% glycerin (Sigma, St. Louis, MO, USA), we used an Olympus BX51 fluorescence microscopy equipped with an Olympus Micro DP72 camera (Olympus Optical Co., Ltd, Tokyo, Japan) to detect the QD signals. The 605-QDs were excited by blue light (excitation wavelength of 450–480 nm) and the specific positive signal was red and photo stable. To validate the accuracy of QDs-IHC results, IHC staining was performed and the results were interpreted and compared to the QDs-IHC results. 2.3. Immunohistochemistry (IHC) The procedures of IHC were proceeded according to the standard streptavidin-peroxidase method as routine. Briefly, 5 μm-thick TMAs slides were deparaffinized in xylene, treated with 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity, microwaved for 15 minutes in citric acid buffer (10 mM, pH 6.0) for antigen retrieval, and followed by incubation with 10% normal goat serum for 30 minutes to block nonspecific binding. The antibodies against PI3K, p-Akt and PTEN (as above) were then applied as the primary anti-
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma
301
Fig. 1. Quantum dots-based immunofluorescent staining of PI3K, p-AKT, and PTEN protein in different histology of small intestinal tissues. A-C show staining of PI3K in small intestinal adenocarcinoma, non-cancer tissue and normal intestinal mucosa, respectively. D-F show staining of p-Akt in small intestinal adenocarcinoma,non-cancer tissue and normal intestinal mucosa, respectively. G-I show staining of PTEN in small intestinal adenocarcinoma, non-cancer tissue and normal intestinal mucosa, respectively (× 100). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130352)
body at the same dilution at QDs-IHC. After washing in TBS, TMAs were incubated in biotinylated goat anti-rabbit IgG, washed again in TBS, and incubated with streptavidin-peroxidase complex (ready-to use, Zymed, South San Francisco, USA) for 15 minutes at 37◦ C. The staining was visualized by reaction with 3, 3’-diaminobenzidine substrate-chromogen solution (Zhongshan Golden Bridge Biotechnology CO, LTD, China) for 1–5 minutes at room temperature and monitored under the microscope, followed by counterstaining with hematoxylin. The immunostained TMAs were reviewed under a light microscope, and both membrane and cytoplasmic stains were considered positive. 2.4. Scoring of QDs-IHC and IHC results We adopted a scoring system based on both the staining intensity and the extent percentage of stained
cells for quantitation of results. Intensity was scored as follows: 0, no staining; 1, weak; 2, moderate; and 3, strong. Extent percentage was scored as 1, 2, 3 if the percentage of the positive cells was less than 25%, 25%∼50%, or greater than 50%, respectively. The IHC and QDs-IHC scores was then defined as the sum of the two scores, specimens were considered positive if the score was greater than 4. 2.5. Statistical analysis The different rates comparison was analyzed using Chi-square and Fisher’s exact tests. The difference between conventional IHC and QDs-IHC was calculated by Mcnemar’s test. The correlation of the QDs-IHC and IHC scores between PI3K and p-Akt in SIA group was calculated by Spearman’s rho correlation test. Statistical analyses were performed using SPSS 13.0 soft-
302
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma Table 1 PI3K, p-Akt and PTEN protein expression in three groups detected by QDs-IHC
Group
n
SIA1 Non-cancer2 Normal small intestine tissue3 Overall
53 11 11 75
PI3K − + PR(%) P 12 41 77.4 1 vs. 2 : 0.015∗ 7 4 36.4 1 vs. 3 : 0.015∗ 7 4 36.4 2 vs. 3 : 1.000 26 49 65.3
p-Akt − + PR(%) P 12 41 77.4 1 vs. 2 : 0.002∗ 8 3 27.3 1 vs. 3 : 0.000∗ 9 2 18.2 2 vs. 3 : 1.000 29 46 61.3
PTEN − + PR(%) P 31 22 41.5 1 vs. 2 : 0.513 5 6 54.5 1 vs. 3 : 1.000 7 4 36.4 2 vs. 3 : 0.670 43 32 42.7
PR: positive rate. ∗ P < 0.05 was considered as statistically significant. Table 2 PI3K, p-Akt and PTEN protein expression in three groups detected by conventional IHC Group
n
SIA1 Non-cancer2 Normal small intestine tissue3 Overall
53 11 11 75
PI3K − + PR(%) P 20 33 62.3 1 vs. 2 : 0.017∗ 9 2 18.2 1 vs. 3 : 0.002∗ 10 1 9.1 2 vs. 3 : 1.000 39 36 48
p-Akt − + PR(%) P 14 39 73.6 1 vs. 2 : 0.000∗ 10 1 9.1 1 vs. 3 : 0.000∗ 11 0 0 2 vs. 3 : 0.000∗ 35 40 53.3
PTEN − + PR(%) P 28 25 47.2 1 vs. 2 : 0.000∗ 11 0 0 1 vs. 3 : 0.714 7 4 36.4 2 vs. 3 : 0.000∗ 46 29 38.6
PR: positive rate. ∗ P < 0.05 was considered as statistically significant.
A
B
C
Fig. 2. Immunohistochemical staining of PI3K, p-Akt, and PTEN protein in the same spot of small intestinal adenocarnoma (× 40). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130352)
ware (SPSS Inc., Chicago, IL, USA) and P < 0.05 was considered statistically significant.
3. Results 3.1. In situ imaging of P I3K, p-Akt and PTEN protein expression in the TMAs Digital images showed QD fluorescent photographs of PI3K, p-Akt and PTEN protein expression in the TMAs (Fig. 1). Immunohistochemical staining was also carried out (Fig. 2). All biomarkers immunoreactivity was assessed by examination of the cytoplasmic staining. The positive signal of QDs-IHC was red and bright while the background signal was green and dark so as to make it easy to distinguish. The positive signal of IHC was dark blue.
3.2. Expression of PI3K, p-Akt and PTEN detected by QDs-IHC and IHC We subgrouped all the cases by the pathological types of the small intestine tissues. The results were summarized in Table 1 and Table 2. The QDs-IHC results showed that the positive rates of PI3K and pAkt expression were significantly higher in SIA group (77.4%, 41/53 and 77.4%, 41/53, respectively) than that in non-cancer group (36.4%, 4/11 and 27.3, 3/11, respectively) and normal tissue group (36.4%, 4/11 and 18.2, 2/11, respectively). However, the difference of the positive rate of the PTEN expression in three groups did not reach a statistical difference (P = 0.761). Most of the IHC results were in concordance with the QDs-IHC. The results of conventional IHC and QDs-IHC were compared in Table 3. There existed a significant differ-
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma
303
Table 3 Comparison of difference between conventional IHC and QDs-IHC
QDs-IHC ∗P
PI3K + − 35 14 1 25
+ −
PI3K
Conventional IHC p-Akt + − p-Akt + 37 9 − 3 26
P
Cancer Biomarkers 13 (2013) 299–305 DOI 10.3233/CBM-130352 IOS Press
Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma detected by quantum dots-based immunofluorescence technology Yi Zhanga,1, Xue Yaoa,1 , Congqing Jiangb , Junqiu Yuec, Jing Guand , Honglei Chenga, Mohammed Hajirashida , Yan Wanga and Lifang Fana,∗ a
Department of Pathology, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, China Department of General Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China c Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei, China d Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China b
Abstract. BACKGROUND: Small intestinal adenocarcinoma (SIA) is often diagnosed at an advanced stage due to its rarity and nonspecific clinical manifestations. Abnormal activation of the PI3K/Akt pathway may lead to various diseases including cancer. In this study, we explored the relationship between those pathways and SIA. OBJECTIVE: To investigate the expression of PI3K, phospho-Akt (p-Akt) and PTEN in SIA to evaluate their clinical significance. METHODS: Expression of PI3K, p-Akt and PTEN biomarkers were examined by quantum dots-based immunofluorescence histochemistry (QDs-IHC) and traditional immunohistochemistry (IHC). There are 53 SIA samples, 11 non-cancer tissue samples and 11 normal small intestine tissues. RESULTS: The positive frequencies of PI3K and p-Akt expression were significantly higher in the SIA group (77.4%, 41/53 and 77.4%, 41/53, respectively) than that in the non-cancer group (36.4%, 4/11 and 27.3, 3/11, respectively) and normal small intestine tissue group (36.4%, 4/11 and 18.2, 2/11, respectively). However, the difference of the positive rates of PTEN expression in three groups did not reach a statistical difference (P = 0.761). The IHC results were in concordance with the QDs-IHC. CONCLUSIONS: These observations support that PI3K/Akt pathway is highly activated in SIA, which may implicate therapeutic targeting of the PI3K/Akt signaling pathway for its treatment. Keywords: Adenocarcinoma, PI3K, PTEN, p-Akt, quantum dots, small intestine
1. Introduction Small intestine accounts for 75% of the length of gastrointestinal tract and over 90% of the mucosal sur1 These
authors contributed equally to this study. author: Lifang Fan, Department of Pathology, School of Basic Medical Science, Wuhan University, 185 Donghu Road, Wuchang District, 430071 Wuhan, Hubei, China. E-mail: [email protected]. ∗ Corresponding
face. But only 2%–3% of malignancies of the gastrointestinal system happen in the small bowel, whereas, colorectal carcinoma is the third most common cancer worldwide. It has been confirmed that SIA showed different immunophenotypes compared with colorectal adenocarcinoma, though SIA is morphologically similar to colorectal adenocarcinoma and shares many of the risk factors in tumorigenesis [1]. Little research has been done into the molecular mechanisms of SIA due to the lack of samples.
c 2013 – IOS Press and the authors. All rights reserved ISSN 1574-0153/13/$27.50
300
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma
PI3K/Akt pathway plays an important role in pathogenesis of many human cancers, and is involved in DNA synthesis and cell proliferation, and PTEN is a major negative regulator of the PI3K/Akt signaling pathway. Abnormal activation of the pathway may lead to various diseases including cancer [2]. However, few studies about this pathway in SIA have been carried out. QDs-IHC is a newly developed technique to detect the expressions of proteins. It can stain both superficial and deeper parts of the tumor cells, and the QD fluorescence of the tumor cell remains unchanged when background tissue autofluorescence changes with the lengths of different excitation waves, which makes it much easier to detect even a very small nest of tumor cells [3]. Previous studies demonstrated that QDsIHC possess advantages of a higher fluorescent efficiency, greater adaptability, higher photostability and longer preservation, making it more sensitive and accurate than conventional immunohistochemistry, especially in the evaluation of low-expressed genes [4,5]. SIA is often diagnosed at an advanced stage due to its rarity and nonspecific clinical manifestations, which contributes to a poor prognosis. Hereby, we performed quantum dots-based immunofluorescence in the current study to investigate the expressions of PI3K, p-Akt and PTEN in samples of SIA patients, in an attempt to help with the clinical treatment of SIA. The results turn out that PI3K and p-Akt proteins are overexpressed in SIA samples.
2. Materials and methods 2.1. Tissue samples Small intestine disease spectrum tissue microarrays (TMAs) were obtained from US Biomax Inc. (http://www.biomax.us/tissue-arrays/Intestine/SM802; lot number SM802). Tissue samples were arranged in 10 columns and 8 rows for a total of 80 individual cores (1.5 mm, 5 μm). 5 non-small intestine samples were excluded, and a total of 75 cores of small intestine specimen were examined in this study. 53 patients were grouped into SIA group, in which 45 cases were diagnosed with primary SIA and 8 cases were metastasized to omentum or lymph nodes. 3 cases with ulcer at small intestine and 8 with chronic inflammation were grouped into non-cancer group. 5 samples of normal small intestine tissue and 6 cancer adjacent normal small intestine tissues were grouped into normal tissue group.
2.2. Quantum dots-based immunofluorescence histochemistry (QDs-IHC) QDs-IHC was performed according to the manufacturer’s instructions (Wuhan Jiayuan Quantum Dot Co, LTD, China). The 5 μm TMAs were deparaffinized in xylene and microwaved for 15 minutes in citric acid buffer (10 mM, pH 6.0) for antigen retrieval. For antibody bindings, TMAs were first incubated in 10% normal goat serum for 30 minutes to block nonspecific binding and then at 4◦ C overnight in primary rabbit anti-Phospho-Akt polyclonal antibody (1:50 dilution, #3787, cell signaling, USA), rabbit anti-PTEN polyclonal antibody (1:200 dilution, #9559, cell signaling, USA) and rabbit anti-PI3 Kinase p110α polyclonal antibody (1:400 dilution, #4249, cell signaling, USA), respectively. TMAs were then washed with TBS (0.5% Tween, 0.1 MTris-base, 0.9% NaCl, and pH 7.6) and incubated in biotinylated goat anti-rabbit IgG (1:100 dilution, Jackson Immuno Research, West Grove, A). Control groups were prepared in parallel but incubated in TBS instead of primary antibody. The TMAs were again washed in TBS and finally incubated with the QDs conjugated streptavidin (QDs-SA) probes (605nm-QDs-SA, cat. no. L-1002-1), prepared by Wuhan Jiayuan Quantum Dots Co., Ltd (Wuhan, China) for 30 min at 37◦ C. After washing in TBS and sealing with 90% glycerin (Sigma, St. Louis, MO, USA), we used an Olympus BX51 fluorescence microscopy equipped with an Olympus Micro DP72 camera (Olympus Optical Co., Ltd, Tokyo, Japan) to detect the QD signals. The 605-QDs were excited by blue light (excitation wavelength of 450–480 nm) and the specific positive signal was red and photo stable. To validate the accuracy of QDs-IHC results, IHC staining was performed and the results were interpreted and compared to the QDs-IHC results. 2.3. Immunohistochemistry (IHC) The procedures of IHC were proceeded according to the standard streptavidin-peroxidase method as routine. Briefly, 5 μm-thick TMAs slides were deparaffinized in xylene, treated with 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity, microwaved for 15 minutes in citric acid buffer (10 mM, pH 6.0) for antigen retrieval, and followed by incubation with 10% normal goat serum for 30 minutes to block nonspecific binding. The antibodies against PI3K, p-Akt and PTEN (as above) were then applied as the primary anti-
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma
301
Fig. 1. Quantum dots-based immunofluorescent staining of PI3K, p-AKT, and PTEN protein in different histology of small intestinal tissues. A-C show staining of PI3K in small intestinal adenocarcinoma, non-cancer tissue and normal intestinal mucosa, respectively. D-F show staining of p-Akt in small intestinal adenocarcinoma,non-cancer tissue and normal intestinal mucosa, respectively. G-I show staining of PTEN in small intestinal adenocarcinoma, non-cancer tissue and normal intestinal mucosa, respectively (× 100). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130352)
body at the same dilution at QDs-IHC. After washing in TBS, TMAs were incubated in biotinylated goat anti-rabbit IgG, washed again in TBS, and incubated with streptavidin-peroxidase complex (ready-to use, Zymed, South San Francisco, USA) for 15 minutes at 37◦ C. The staining was visualized by reaction with 3, 3’-diaminobenzidine substrate-chromogen solution (Zhongshan Golden Bridge Biotechnology CO, LTD, China) for 1–5 minutes at room temperature and monitored under the microscope, followed by counterstaining with hematoxylin. The immunostained TMAs were reviewed under a light microscope, and both membrane and cytoplasmic stains were considered positive. 2.4. Scoring of QDs-IHC and IHC results We adopted a scoring system based on both the staining intensity and the extent percentage of stained
cells for quantitation of results. Intensity was scored as follows: 0, no staining; 1, weak; 2, moderate; and 3, strong. Extent percentage was scored as 1, 2, 3 if the percentage of the positive cells was less than 25%, 25%∼50%, or greater than 50%, respectively. The IHC and QDs-IHC scores was then defined as the sum of the two scores, specimens were considered positive if the score was greater than 4. 2.5. Statistical analysis The different rates comparison was analyzed using Chi-square and Fisher’s exact tests. The difference between conventional IHC and QDs-IHC was calculated by Mcnemar’s test. The correlation of the QDs-IHC and IHC scores between PI3K and p-Akt in SIA group was calculated by Spearman’s rho correlation test. Statistical analyses were performed using SPSS 13.0 soft-
302
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma Table 1 PI3K, p-Akt and PTEN protein expression in three groups detected by QDs-IHC
Group
n
SIA1 Non-cancer2 Normal small intestine tissue3 Overall
53 11 11 75
PI3K − + PR(%) P 12 41 77.4 1 vs. 2 : 0.015∗ 7 4 36.4 1 vs. 3 : 0.015∗ 7 4 36.4 2 vs. 3 : 1.000 26 49 65.3
p-Akt − + PR(%) P 12 41 77.4 1 vs. 2 : 0.002∗ 8 3 27.3 1 vs. 3 : 0.000∗ 9 2 18.2 2 vs. 3 : 1.000 29 46 61.3
PTEN − + PR(%) P 31 22 41.5 1 vs. 2 : 0.513 5 6 54.5 1 vs. 3 : 1.000 7 4 36.4 2 vs. 3 : 0.670 43 32 42.7
PR: positive rate. ∗ P < 0.05 was considered as statistically significant. Table 2 PI3K, p-Akt and PTEN protein expression in three groups detected by conventional IHC Group
n
SIA1 Non-cancer2 Normal small intestine tissue3 Overall
53 11 11 75
PI3K − + PR(%) P 20 33 62.3 1 vs. 2 : 0.017∗ 9 2 18.2 1 vs. 3 : 0.002∗ 10 1 9.1 2 vs. 3 : 1.000 39 36 48
p-Akt − + PR(%) P 14 39 73.6 1 vs. 2 : 0.000∗ 10 1 9.1 1 vs. 3 : 0.000∗ 11 0 0 2 vs. 3 : 0.000∗ 35 40 53.3
PTEN − + PR(%) P 28 25 47.2 1 vs. 2 : 0.000∗ 11 0 0 1 vs. 3 : 0.714 7 4 36.4 2 vs. 3 : 0.000∗ 46 29 38.6
PR: positive rate. ∗ P < 0.05 was considered as statistically significant.
A
B
C
Fig. 2. Immunohistochemical staining of PI3K, p-Akt, and PTEN protein in the same spot of small intestinal adenocarnoma (× 40). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130352)
ware (SPSS Inc., Chicago, IL, USA) and P < 0.05 was considered statistically significant.
3. Results 3.1. In situ imaging of P I3K, p-Akt and PTEN protein expression in the TMAs Digital images showed QD fluorescent photographs of PI3K, p-Akt and PTEN protein expression in the TMAs (Fig. 1). Immunohistochemical staining was also carried out (Fig. 2). All biomarkers immunoreactivity was assessed by examination of the cytoplasmic staining. The positive signal of QDs-IHC was red and bright while the background signal was green and dark so as to make it easy to distinguish. The positive signal of IHC was dark blue.
3.2. Expression of PI3K, p-Akt and PTEN detected by QDs-IHC and IHC We subgrouped all the cases by the pathological types of the small intestine tissues. The results were summarized in Table 1 and Table 2. The QDs-IHC results showed that the positive rates of PI3K and pAkt expression were significantly higher in SIA group (77.4%, 41/53 and 77.4%, 41/53, respectively) than that in non-cancer group (36.4%, 4/11 and 27.3, 3/11, respectively) and normal tissue group (36.4%, 4/11 and 18.2, 2/11, respectively). However, the difference of the positive rate of the PTEN expression in three groups did not reach a statistical difference (P = 0.761). Most of the IHC results were in concordance with the QDs-IHC. The results of conventional IHC and QDs-IHC were compared in Table 3. There existed a significant differ-
Y. Zhang et al. / Expression of PI3K, PTEN and Akt in small intestinal adenocarcinoma
303
Table 3 Comparison of difference between conventional IHC and QDs-IHC
QDs-IHC ∗P
PI3K + − 35 14 1 25
+ −
PI3K
Conventional IHC p-Akt + − p-Akt + 37 9 − 3 26
P
