Med Oncol (2014) 31:983 DOI 10.1007/s12032-014-0983-7
ORIGINAL PAPER
Clinical significance of PIK3CA and survivin in primary adenosquamous lung carcinoma Shaomin Yu • Zhihong Zhang • Bin Zhang Yongqian Shu • Hao Wu • Xiang Huang • Qianqian Yu • Renhua Guo
•
Received: 24 January 2014 / Accepted: 25 April 2014 / Published online: 17 May 2014 Ó Springer Science+Business Media New York 2014
Abstract The aim of this study was to evaluate the expression of PIK3CA and survivin in adenosquamous lung cancer (ASC) patients and their clinicopathological significance. A total of 32 patients with adenosquamous carcinoma and ten cases of normal lung lesion were investigated, and the expressions of PIK3CA and survivin were detected by immunohistochemistry. The PIK3CA and survivin expression in adenosquamous lung carcinoma tissues was significantly higher than those of the normal lesions (p = 0.02). The positive rate of PIK3CA and survivin expressions was in accordance with the tumor-nodemetastasis stage (p = 0.002/0.013), pathological grade (p = 0.019/0.013), and lymph node metastasis (p = 0.029/ 0.008). Out of 15 patients with definite follow-ups, highly positive expressions of PIK3CA (12 cases) and survivin (11 cases) were suggested to be associated with adverse prognosis (nine cases recurrence and four cases died). A positive correlation was also observed between the expressions of PIK3CA and survivin (r = 0.622, Shaomin Yu and Zhihong Zhang have contributed equally to this work. S. Yu Y. Shu H. Wu X. Huang Q. Yu R. Guo (&) Department of Medical Oncology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing 210029, Jiangsu, People’s Republic of China e-mail: [email protected] Z. Zhang Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China B. Zhang Department of Thoracic Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China
p \ 0.001). These findings indicated that PIK3CA and survivin up-regulation might play an important role in lymph node metastasis and adverse prognosis in ASC. Nevertheless, further additional prospective studies in the area of ASC molecular profiling are needed. Keywords Adenosquamous lung carcinoma PIK3CA Survivin Immunohistochemistry
Introduction Primary adenosquamous carcinoma of the lung (ASC) is an uncommon subtype of NSCLC with an aggressive entity. According to the World Health Organization (WHO) histological criteria, ASC is identified as a tumor representing the components of both adenocarcinoma (AC) and squamous carcinoma (SC), with each comprising at least 10 % of the whole tumor [1]. Nevertheless, morphology alone might not be able to differentiate all the cases of ASC, especially for the poorly differentiated cells, since its heterogenic nature frequently leads to a difficult identification of the clinicopathological characteristics. Meanwhile, the prognosis for the patients with ASC remains unsatisfactory. Therefore, more work is needed in the molecular pathogenesis in order to develop further preventive or therapeutic treatments against ASC. Although a number of manuscripts concerning the molecular profiles of the AC and squamous lung carcinoma have been published in the recent years, few papers are available on ASC, especially for the relationship of the molecular profiles on the tumor recurrence (including distant metastasis), which might remain unclarified due to the infrequent occurrence of this type of tumor. In the present study, we investigated the expression of PIK3CA and survivin in both ASC and matched non-cancerous lung
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Med Oncol (2014) 31:983
Materials and methods
Affiliated Hospital of Nanjing Medical University from March 2006 to January 2013. During this period of time, 65 patients were diagnosed with pathologic stage I–IV of adenosquamous lung carcinoma through a routine pathological diagnosis. The cases, which were diagnosed after the biopsy and the ones with a poor immunohistochemistry (IHC) staining, were excluded. A total of 32 patients who underwent the radical surgical resection were selected; however, 17 cases were lost to follow-ups. The follow-ups continued until December 2012. A total of ten cases with benign pulmonary disease specimens were included as normal controls. Patients were free from any chemotherapy, radiotherapy, or any other form of treatment before being sampled. Informed consents were obtained from all the patients or their relatives. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. A total of 23 men and 9 women were included in this study. The age distribution ranged from 43 to 82 years old with a mean age of 60.1 years. The histological classification, grading, and pathological staging of the adenosquamous lung carcinoma were assigned according to the seventh edition of the tumor-node-metastasis (TNM) classification of malignant tumors [6]. Therefore, twelve, eight, eleven, and one cases were in stage I, II, III, and IV (isolated brain metastasis), respectively. Lymph node metastasis was found in 20 cases. Based on the microscopic examination of the HE stained slides, we confirmed the diagnosis of adenosquamous carcinoma (i.e., that the squamous cells and glandular cells of the tumor components both accounted for more than 10 % [1]).
Patients and tumors
Immunohistochemical staining
The clinical records of the patients with adenosquamous lung carcinoma were retrospectively reviewed in the First
The IHC staining was conducted for the primary tumor tissues in this study. For each case, the cross sections of 4
tissues. Meanwhile, the correlations between PIK3CA and survivin expression with the clinicopathological characteristics were also investigated. PI3K is a member of the conserved lipid kinase family that phosphorylates the 3-hydroxyl group of the phosphoinositides, which is associated with tumor initiation, growth, and proliferation [2]. PI3K is a heterodimer consisting of a p85 regulatory and a p110 catalytic subunit. One of the most frequent genetic alterations in this pathway is the amplification of the PI3K catalytic agent, alpha polypeptide (PIK3CA) gene [3]. In our previous research, the expression of PIK3CA in the cancer and the adjacent tissues was significantly higher than that of the normal tissues. Moreover, a higher expression of PIK3CA and survivin was closely associated with the carcinogenesis and metastasis in non-small-cell lung cancer (NSCLC). Survivin was confirmed as a novel member of the inhibitor of apoptosis proteins (IAP) family. It was predominantly expressed during mitosis and anaphase, which directly inhibited the activation of caspase-3 and -7. It was reported that survivin exerted the anti-apoptotic effects and regulated the cell division in the tumor or normal tissues [4]. During tumorigenesis, survivin expression is inversely correlated with the apoptosis inhibition and is positively correlated with the proliferation and angiogenesis [5]. Thereby, in the present work, we focused on the PIK3CA and survivin expression in adenosquamous lung carcinoma and its potential association with the development of the ASC.
Fig. 1 PIK3CA and survivin immunohistochemistry. Negative stain (score 0) in ASC (a, e); 1 ? positive stain in ASC (b, f); 2 ? positive stain in ASC (c, g); 3 ? positive stain in ASC (d, h); and original magnification (a–h, 1009)
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lm thickness were fixed in 10 % formalin and were embedded in paraffin. After the initial deparaffinization, the sections were applied to the routine immunohistochemical staining. Before the IHC staining, the slices were deparaffinized, and the PIK3CA and survivin antibodies were applied and placed in special boxes containing citrate buffer solution (4 mm, pH 6). They were then boiled thrice for 5 min in a microwave oven. The slides were incubated with PIK3CA rabbit polyclonal anti-human antibody (1:100 dilution, Cell Signaling Technology Company) and survivin goat polyclonal anti-human antibody (1:100 dilution, Cell Signaling Technology Company). The slides were incubated with a general secondary antibody and horseradish peroxidase. The tissues were stained with freshly prepared 3, 3-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated, and mounted. The first antibody was substituted with phosphatebuffered saline as the negative control. Meanwhile, an already-known positive tissue was selected as a positive control. All the slides were separately verified by the same technician in the same laboratory. Assessment of each slide was done by two independent pathologists, who were unaware of the clinical details of the patients. Different opinions were resolved by a joint review together with a senior pathologist. Evaluation of the immunoreactivity for PIK3CA and survivin was analyzed via using a semiquantitative scoring system, which was based on the intensity and the distribution of the cytoplasmic stain. Therefore, tumors were considered to be positive when the cells demonstrated unequivocal cytoplasmic staining, showing the clear expression of brown particles. Briefly, after immunostaining with anti-PIK3CA and survivin antibodies, the stained sections were screened at an original magnification to evaluate the regions of the highest PIK3CA and survivin expression. They were randomly selected and were determined in different fields of serial sections. Based on the distribution of the immunoreactivity in the cytoplasm of the tumor cells, the IHC staining was scored as focal (\15 %; 1 point), regional (15–50 %; 2 points), and diffuse ([50 %; 3 points). The intensity of cytoplasmic staining was graded as weak (1 point), moderate (2 points), or intense (3 points) in comparison with the negative controls (0 point). According to the SxC formulation, the sections with a score of less than four were
Table 1 PIK3CA and Survivn expression in ASC and normal controls
considered as negative, otherwise they were considered as positive. Statistical analysis Statistical analyses were conducted using v2 test and Fisher’s exact test in order to determine the expression of PIK3CA and survivin among different clinicopathological parameters. To assess the relationship between PIK3CA and survivin expression, Spearman’s correlation analysis was performed. All the outcomes were recorded into SPSS 13.0 statistical software program. Results were considered statistically significant for two-tailed analysis when the p value was less than 0.05.
Results PIK3CA and survivin were specifically expressed in the human ASC tissues As depicted in Fig. 1a, e was normal controls, corresponding to the expression of PIK3CA and survivin. Figure 1b, f is stained in light yellow in comparison with c and g, which were stained in a brownish yellow. Furthermore, there was a sharp increase in the color intensity of the Fig. 1d, h, where they were stained in sepia. Out of 32 patients with ASC, the positive immunostaining ratio for PIK3CA and survivin was 78.75 % (25/32 cases) and 68.75 % (22/32 cases), respectively. Whereas in normal tissues, the positive rates of PIK3CA and survivin expressions were detected in 2 (20.00 %) and 1 (10.00 %) cases, respectively. These data indicated that ASC tissues marked by PIK3CA and survivin were significantly higher than those in the benign lesions (p = 0.02 \ 0.05; Table 1). The expression of PIK3CA and survivin in ASC and their relationships with the clinicopathological characteristics In our study, PIK3CA and survivin expressions were not significantly correlated with the gender (0.657/0.681), age (0.254/0.265), smoking history (0.669/0.712), and tumor size (0.649/0.636) as demonstrated based on the correlation
PIK3CA (?) N Tumor(n = 32) Normal control (n = 10)
Survivin (?)
Positive rate (%)
p value (%)
N
Positive rate (%)
p value (%)
25
78.13
0.02
2
20.00
22
68.75
0.02
1
10.00
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Med Oncol (2014) 31:983
Table 2 PIK3CA and survivin expression in ASC and clinicopathological character
Table 3 PIK3CA and survivin expression in 15 patients with definite follow-ups
Factor
Recurrence
PIK3CA
Survivin
6
?
?
1
-
?
2
?
-
?
?
-
-
PIK3CA (?) N
Positive rate (%)
Survivin (?) p value (%)
N
Positive rate (%)
p value (%)
Sex F
18
78.3
M
7
77.8
[0.05
15
65.2
7
77.8
0.681
Death 4 SD
Age C60
12
70.6
\60
13
86.7
0.402
10
58.8
12
80.0
13
72.2
9
64.3
0.265
2
0.712
group 2, tissues were positive for PIK3CA but negative for survivin. Group 3 was positive for survivin, while negative for PIK3CA. In group 4, neither of the two proteins was detected. The correlation analysis indicated that the positivity for PIK3CA was significantly associated with the positive expression of survivin (r = 0.622, p \ 0.001).
Smoking history Yes
15
83.3
No
10
71.4
[0.05
Tumor size (cm) C5
11
78.6
\5
14
77.8
0.649
9
69.2
13
68.4
3
33.3
19
82.6
[0.05
Histological grade Well and moderate Poor
5
50.0
20
90.9
0.019
0.013
Discussion
TNM stage II
13
65.0
III–IV
12
100.0
0.029
12
54.5
10
100.0
0.013
Lymph node metastasis 0
13
65.0
C1
12
100.0
0.029
16
88.9
6
42.9
0.008
analysis (p [ 0.05). However, the tissues marked by PIK3CA and survivin were closely linked to the histological grade (0.019/0.013), TNM stage (0.002/0.013), and lymph node metastasis (0.029/0.008) as demonstrated in Table 2. Out of the 32 cases, there were 15 patients with definite follow-ups. A total of nine patients with lymph node metastasis died due to recurrence, of which six cases showed positive expression of both PIK3CA and survivin, and three cases showed positive expression of either PIK3CA or survivin. Furthermore, the four cases that died within 1 year of the surgical resection were all positive for PIK3CA and survivin expression. Two cases achieved stable disease (SD) after the surgery, and both displayed negative expression of PIK3CA and survivin (Table 3). The relationships between PIK3CA and survivin expression The relationship between PIK3CA and survivin expression was studied among 32 ASC patients. Our results showed that ASC could be classified into four groups. In group 1, PIK3CA and survivin demonstrated positive staining. In
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The incidence of adenosquamous lung carcinoma varies from 0.4 to 4 % of all lung cancers according to the WHO Classification of Tumors, which was published in 2004 [1]. This low incidence of ASC might be attributed to three main factors. First, the preoperative biopsy specimen often leads to removal of the AC or only the SCC component, which cannot be easily found. Secondly, it is difficult to histologically classify the ASC because of the undifferentiated cells. Finally, there are no studies with adequate size of patient population for a definitive statistical analysis of the nature of ASC. Heterogeneity is a kind of character, which frequently occurs in cancer. ASC is an atypical heterogeneous tumor including two distinct components of AC and SC. When it comes to the histogenesis of ASC, there are many hypotheses, including AC with squamous metaplasia, collision tumor, high-grade mucoepidermoid carcinoma, and bipotential undifferentiated cell origin [7]. Niho and colleagues [8] reported that SC and AC components showed identical monoclonal patterns, which suggested that both of these components originated from the same cell type. Kanazawa et al. [9] also suggested the monoclonal transition from SC to AC in the carcinogenic process. The PI3K activation is commonly observed in many human cancer cells. While survivin expression is elevated in cancer cells, it is further induced by some growth factors through PI3K activation. The PI3K/Akt signaling pathway has been implicated in the up-regulation of survivin in both vascular endothelial and tumor cells [10]. Consequently, in our study, we explored whether PIK3CA and survivin
Med Oncol (2014) 31:983
expressions were up-regulated in ASC. Many studies mentioned that a high lymph node metastasis might lead to an adverse prognosis. Filosso and colleagues reported that an aggressive biological behavior of ASC was demonstrated by a high lymph node metastases percentage at presentation (29 patients, 60.4 %), which could translate into a higher pathological stage at the intervention (ASC stages II, III, and IV: 33/48, p = 0.0001) [11]. Ichiro and colleagues reported that in comparison with the negative staining group, the presence of node metastasis was significantly higher in the group that stained positive for PIK3CA in esophageal squamous cell carcinoma [12]. Li et al. [13] pointed out that a parallel molecular detection of survivin and livin mRNAs was a specific diagnostic tool for the assessment of lymph node micrometastasis, which could show powerful prognostic implications for the patients with a completely resected stage I NSCLC. As for our study, the outcome revealed that the positive rate of PIK3CA and survivin expressions in ASC had a remarkable relationship with the lymph node metastasis, which was in line with the above-mentioned reports. The range of the lymph node metastasis was a vital prognostic factor for the patients. As the lung ASC has an infrequent occurrence and is very complex, an early diagnosis of the lymph node metastasis is difficult but necessary. Taken this into the consideration, a high expression of PIK3CA and survivin in the adenosquamous lung carcinoma might act as a predictor for lymph node metastasis. The biological behavior and clinicopathological characteristics of ASC have not yet been thoroughly unraveled. Among the 15 patients with follow-ups, there were 9 patients with lymph node metastasis (either PIK3CA- or survivin-positive expression) that died due to recurrence, such as brain and bone distant metastasis. Furthermore, four cases died within 1 year of the surgical resection, and all of them were positive for PIK3CA and survivin expression. These results suggested that positive expression of PIK3CA and survivin was associated with adverse prognosis, especially for double positivity. Alternatively, double-negative expression of PIK3CA and survivin might suggest a good prognosis. The similar results were also observed in Lin’s study that demonstrated an overexpression of PI3K might be linked to the process of tumor-cell transformation in lung carcinomas, including regional lymph nodes metastasis and the occurrence of distant metastases [14]. In the series by Guo and colleagues, they found that the expression of survivin and p-Akt was positively correlated to the tumor recurrence in the HCC patients. With regard to our study, there might be a potential tendency toward the relationship between the expression of PIK3CA and survivin with recurrence. However, there were still some weak points in this paper. To our knowledge, IHC staining has often been used
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for semi-quantitative measurement of the proteins expressed in the tissues. However, this method might be limited by variation according to the different antibodies used. Therefore, other methods should further support the results in the present study, such as real-time PCR and Western blot. Moreover, more patients and follow-ups will be required. In summary, we suggested that the expression of PIK3CA and survivin had a significant correlation with the TNM stage, pathological grade, and lymph node metastasis. Further clinical studies should be carried out on the patients with adenosquamous lung carcinoma in order to determine the relationships between the PIK3CA- and survivin-positive expression and recurrence. PIK3CA and survivin might be suggested as potential targets for the development of therapeutic strategies for adenosquamous lung carcinoma. Acknowledgments This work was supported by grants from the National Nature Science Foundation of China (81071228), Medical Important Talents of Jiangsu Province (RC201157), and Project of Oncology Translational Medicine Central of Jiangsu Province (BL2012008). Conflict of interest financial one.
There was no conflict of interest including any
References 1. Travis W, Brambilla E, Muller-Hermelink H, Harris C. Tumours of the lung, pleura, thymus and heart. Pathology and genetics. World Health Organization Classification of Tumours. Lyon: IARC Press; 2004. 2. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene. 2008;27(41):5497–510. doi:10. 1038/onc.2008.245. 3. De Luca A, Maiello MR, D’Alessio A, Pergameno M, Normanno N. The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets. 2012;16(S2): S17–27. 4. Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res. 2008;14(16):5000–5. 5. Yamamoto T, Tanigawa N. The role of survivin as a new target of diagnosis and treatment in human cancer. Med Electron Microsc. 2001;34(4):207–12. 6. Goldstraw P. Site-specific explanatory notes for lung and pleural tumours. International Association for the Study of Lung Cancer Staging Handbook in Thoracic Oncology. Orange Park: Editorial Rx Press; 2009. p. 53–74. 7. Ichinose Y, Hara N, Takamori S, Maeda K, Yano T, Ohta M. DNA ploidy pattern of each carcinomatous component in adenosquamous lung carcinoma. Ann Thorac Surg. 1993;55(3): 593–6. 8. Niho S, Yokose T, Kodama T, Nishiwaki Y, Mukai K. Clonal analysis of adenosquamous carcinoma of the lung. Cancer Sci. 1999;90(11):1244–7. 9. Kanazawa H, Ebina M, Ino-oka N, Shimizukawa M, Takahashi T, Fujimura S, et al. Transition from squamous cell carcinoma to
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983 Page 6 of 6 adenocarcinoma in adenosquamous carcinoma of the lung. Am J Pathol. 2000;156(4):1289–98. 10. Zhao P, Meng Q, Liu LZ, You YP, Liu N, Jiang BH. Regulation of survivin by PI3K/Akt/p70S6K1 pathway. Biochem Biophys Res Commun. 2010;395(2):219–24. doi:10.1016/j.bbrc.2010.03. 165. 11. Maeda H, Matsumura A, Kawabata T, Suito T, Kawashima O, Watanabe T, et al. Adenosquamous carcinoma of the lung: surgical results as compared with squamous cell and adenocarcinoma cases. Eur J Cardio-thorac Surg Off J Eur Assoc Cardiothorac Surg. 2012;41(2):357–61. doi:10.1016/j.ejcts.2011.05.050. 12. Akagi I, Miyashita M, Makino H, Nomura T, Hagiwara N, Takahashi K, et al. Overexpression of PIK3CA is associated with
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Med Oncol (2014) 31:983 lymph node metastasis in esophageal squamous cell carcinoma. Int J Oncol. 2009;34(3):767. 13. Li J, Li ZN, Yu LC, Shi SB, Ge LP, Wu JR, et al. Gene diagnosis of micrometastases in regional lymph nodes of patients with stage I non-small cell lung cancer: impact on staging and prognosis. Clin Transl Oncol Off Publ Fed Span Oncol Soc Natl Cancer Inst Mexico. 2013;. doi:10.1007/s12094-013-1017-1. 14. Lin X, Bohle AS, Dohrmann P, Leuschner I, Schulz A, Kremer B, et al. Overexpression of phosphatidylinositol 3-kinase in human lung cancer. Langenbeck’s Arch Surg. 2001;386(4):293–301.
ORIGINAL PAPER
Clinical significance of PIK3CA and survivin in primary adenosquamous lung carcinoma Shaomin Yu • Zhihong Zhang • Bin Zhang Yongqian Shu • Hao Wu • Xiang Huang • Qianqian Yu • Renhua Guo
•
Received: 24 January 2014 / Accepted: 25 April 2014 / Published online: 17 May 2014 Ó Springer Science+Business Media New York 2014
Abstract The aim of this study was to evaluate the expression of PIK3CA and survivin in adenosquamous lung cancer (ASC) patients and their clinicopathological significance. A total of 32 patients with adenosquamous carcinoma and ten cases of normal lung lesion were investigated, and the expressions of PIK3CA and survivin were detected by immunohistochemistry. The PIK3CA and survivin expression in adenosquamous lung carcinoma tissues was significantly higher than those of the normal lesions (p = 0.02). The positive rate of PIK3CA and survivin expressions was in accordance with the tumor-nodemetastasis stage (p = 0.002/0.013), pathological grade (p = 0.019/0.013), and lymph node metastasis (p = 0.029/ 0.008). Out of 15 patients with definite follow-ups, highly positive expressions of PIK3CA (12 cases) and survivin (11 cases) were suggested to be associated with adverse prognosis (nine cases recurrence and four cases died). A positive correlation was also observed between the expressions of PIK3CA and survivin (r = 0.622, Shaomin Yu and Zhihong Zhang have contributed equally to this work. S. Yu Y. Shu H. Wu X. Huang Q. Yu R. Guo (&) Department of Medical Oncology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Rd, Nanjing 210029, Jiangsu, People’s Republic of China e-mail: [email protected] Z. Zhang Department of Pathology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China B. Zhang Department of Thoracic Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China
p \ 0.001). These findings indicated that PIK3CA and survivin up-regulation might play an important role in lymph node metastasis and adverse prognosis in ASC. Nevertheless, further additional prospective studies in the area of ASC molecular profiling are needed. Keywords Adenosquamous lung carcinoma PIK3CA Survivin Immunohistochemistry
Introduction Primary adenosquamous carcinoma of the lung (ASC) is an uncommon subtype of NSCLC with an aggressive entity. According to the World Health Organization (WHO) histological criteria, ASC is identified as a tumor representing the components of both adenocarcinoma (AC) and squamous carcinoma (SC), with each comprising at least 10 % of the whole tumor [1]. Nevertheless, morphology alone might not be able to differentiate all the cases of ASC, especially for the poorly differentiated cells, since its heterogenic nature frequently leads to a difficult identification of the clinicopathological characteristics. Meanwhile, the prognosis for the patients with ASC remains unsatisfactory. Therefore, more work is needed in the molecular pathogenesis in order to develop further preventive or therapeutic treatments against ASC. Although a number of manuscripts concerning the molecular profiles of the AC and squamous lung carcinoma have been published in the recent years, few papers are available on ASC, especially for the relationship of the molecular profiles on the tumor recurrence (including distant metastasis), which might remain unclarified due to the infrequent occurrence of this type of tumor. In the present study, we investigated the expression of PIK3CA and survivin in both ASC and matched non-cancerous lung
123
983 Page 2 of 6
Med Oncol (2014) 31:983
Materials and methods
Affiliated Hospital of Nanjing Medical University from March 2006 to January 2013. During this period of time, 65 patients were diagnosed with pathologic stage I–IV of adenosquamous lung carcinoma through a routine pathological diagnosis. The cases, which were diagnosed after the biopsy and the ones with a poor immunohistochemistry (IHC) staining, were excluded. A total of 32 patients who underwent the radical surgical resection were selected; however, 17 cases were lost to follow-ups. The follow-ups continued until December 2012. A total of ten cases with benign pulmonary disease specimens were included as normal controls. Patients were free from any chemotherapy, radiotherapy, or any other form of treatment before being sampled. Informed consents were obtained from all the patients or their relatives. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. A total of 23 men and 9 women were included in this study. The age distribution ranged from 43 to 82 years old with a mean age of 60.1 years. The histological classification, grading, and pathological staging of the adenosquamous lung carcinoma were assigned according to the seventh edition of the tumor-node-metastasis (TNM) classification of malignant tumors [6]. Therefore, twelve, eight, eleven, and one cases were in stage I, II, III, and IV (isolated brain metastasis), respectively. Lymph node metastasis was found in 20 cases. Based on the microscopic examination of the HE stained slides, we confirmed the diagnosis of adenosquamous carcinoma (i.e., that the squamous cells and glandular cells of the tumor components both accounted for more than 10 % [1]).
Patients and tumors
Immunohistochemical staining
The clinical records of the patients with adenosquamous lung carcinoma were retrospectively reviewed in the First
The IHC staining was conducted for the primary tumor tissues in this study. For each case, the cross sections of 4
tissues. Meanwhile, the correlations between PIK3CA and survivin expression with the clinicopathological characteristics were also investigated. PI3K is a member of the conserved lipid kinase family that phosphorylates the 3-hydroxyl group of the phosphoinositides, which is associated with tumor initiation, growth, and proliferation [2]. PI3K is a heterodimer consisting of a p85 regulatory and a p110 catalytic subunit. One of the most frequent genetic alterations in this pathway is the amplification of the PI3K catalytic agent, alpha polypeptide (PIK3CA) gene [3]. In our previous research, the expression of PIK3CA in the cancer and the adjacent tissues was significantly higher than that of the normal tissues. Moreover, a higher expression of PIK3CA and survivin was closely associated with the carcinogenesis and metastasis in non-small-cell lung cancer (NSCLC). Survivin was confirmed as a novel member of the inhibitor of apoptosis proteins (IAP) family. It was predominantly expressed during mitosis and anaphase, which directly inhibited the activation of caspase-3 and -7. It was reported that survivin exerted the anti-apoptotic effects and regulated the cell division in the tumor or normal tissues [4]. During tumorigenesis, survivin expression is inversely correlated with the apoptosis inhibition and is positively correlated with the proliferation and angiogenesis [5]. Thereby, in the present work, we focused on the PIK3CA and survivin expression in adenosquamous lung carcinoma and its potential association with the development of the ASC.
Fig. 1 PIK3CA and survivin immunohistochemistry. Negative stain (score 0) in ASC (a, e); 1 ? positive stain in ASC (b, f); 2 ? positive stain in ASC (c, g); 3 ? positive stain in ASC (d, h); and original magnification (a–h, 1009)
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Med Oncol (2014) 31:983
Page 3 of 6 983
lm thickness were fixed in 10 % formalin and were embedded in paraffin. After the initial deparaffinization, the sections were applied to the routine immunohistochemical staining. Before the IHC staining, the slices were deparaffinized, and the PIK3CA and survivin antibodies were applied and placed in special boxes containing citrate buffer solution (4 mm, pH 6). They were then boiled thrice for 5 min in a microwave oven. The slides were incubated with PIK3CA rabbit polyclonal anti-human antibody (1:100 dilution, Cell Signaling Technology Company) and survivin goat polyclonal anti-human antibody (1:100 dilution, Cell Signaling Technology Company). The slides were incubated with a general secondary antibody and horseradish peroxidase. The tissues were stained with freshly prepared 3, 3-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, dehydrated, and mounted. The first antibody was substituted with phosphatebuffered saline as the negative control. Meanwhile, an already-known positive tissue was selected as a positive control. All the slides were separately verified by the same technician in the same laboratory. Assessment of each slide was done by two independent pathologists, who were unaware of the clinical details of the patients. Different opinions were resolved by a joint review together with a senior pathologist. Evaluation of the immunoreactivity for PIK3CA and survivin was analyzed via using a semiquantitative scoring system, which was based on the intensity and the distribution of the cytoplasmic stain. Therefore, tumors were considered to be positive when the cells demonstrated unequivocal cytoplasmic staining, showing the clear expression of brown particles. Briefly, after immunostaining with anti-PIK3CA and survivin antibodies, the stained sections were screened at an original magnification to evaluate the regions of the highest PIK3CA and survivin expression. They were randomly selected and were determined in different fields of serial sections. Based on the distribution of the immunoreactivity in the cytoplasm of the tumor cells, the IHC staining was scored as focal (\15 %; 1 point), regional (15–50 %; 2 points), and diffuse ([50 %; 3 points). The intensity of cytoplasmic staining was graded as weak (1 point), moderate (2 points), or intense (3 points) in comparison with the negative controls (0 point). According to the SxC formulation, the sections with a score of less than four were
Table 1 PIK3CA and Survivn expression in ASC and normal controls
considered as negative, otherwise they were considered as positive. Statistical analysis Statistical analyses were conducted using v2 test and Fisher’s exact test in order to determine the expression of PIK3CA and survivin among different clinicopathological parameters. To assess the relationship between PIK3CA and survivin expression, Spearman’s correlation analysis was performed. All the outcomes were recorded into SPSS 13.0 statistical software program. Results were considered statistically significant for two-tailed analysis when the p value was less than 0.05.
Results PIK3CA and survivin were specifically expressed in the human ASC tissues As depicted in Fig. 1a, e was normal controls, corresponding to the expression of PIK3CA and survivin. Figure 1b, f is stained in light yellow in comparison with c and g, which were stained in a brownish yellow. Furthermore, there was a sharp increase in the color intensity of the Fig. 1d, h, where they were stained in sepia. Out of 32 patients with ASC, the positive immunostaining ratio for PIK3CA and survivin was 78.75 % (25/32 cases) and 68.75 % (22/32 cases), respectively. Whereas in normal tissues, the positive rates of PIK3CA and survivin expressions were detected in 2 (20.00 %) and 1 (10.00 %) cases, respectively. These data indicated that ASC tissues marked by PIK3CA and survivin were significantly higher than those in the benign lesions (p = 0.02 \ 0.05; Table 1). The expression of PIK3CA and survivin in ASC and their relationships with the clinicopathological characteristics In our study, PIK3CA and survivin expressions were not significantly correlated with the gender (0.657/0.681), age (0.254/0.265), smoking history (0.669/0.712), and tumor size (0.649/0.636) as demonstrated based on the correlation
PIK3CA (?) N Tumor(n = 32) Normal control (n = 10)
Survivin (?)
Positive rate (%)
p value (%)
N
Positive rate (%)
p value (%)
25
78.13
0.02
2
20.00
22
68.75
0.02
1
10.00
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Med Oncol (2014) 31:983
Table 2 PIK3CA and survivin expression in ASC and clinicopathological character
Table 3 PIK3CA and survivin expression in 15 patients with definite follow-ups
Factor
Recurrence
PIK3CA
Survivin
6
?
?
1
-
?
2
?
-
?
?
-
-
PIK3CA (?) N
Positive rate (%)
Survivin (?) p value (%)
N
Positive rate (%)
p value (%)
Sex F
18
78.3
M
7
77.8
[0.05
15
65.2
7
77.8
0.681
Death 4 SD
Age C60
12
70.6
\60
13
86.7
0.402
10
58.8
12
80.0
13
72.2
9
64.3
0.265
2
0.712
group 2, tissues were positive for PIK3CA but negative for survivin. Group 3 was positive for survivin, while negative for PIK3CA. In group 4, neither of the two proteins was detected. The correlation analysis indicated that the positivity for PIK3CA was significantly associated with the positive expression of survivin (r = 0.622, p \ 0.001).
Smoking history Yes
15
83.3
No
10
71.4
[0.05
Tumor size (cm) C5
11
78.6
\5
14
77.8
0.649
9
69.2
13
68.4
3
33.3
19
82.6
[0.05
Histological grade Well and moderate Poor
5
50.0
20
90.9
0.019
0.013
Discussion
TNM stage II
13
65.0
III–IV
12
100.0
0.029
12
54.5
10
100.0
0.013
Lymph node metastasis 0
13
65.0
C1
12
100.0
0.029
16
88.9
6
42.9
0.008
analysis (p [ 0.05). However, the tissues marked by PIK3CA and survivin were closely linked to the histological grade (0.019/0.013), TNM stage (0.002/0.013), and lymph node metastasis (0.029/0.008) as demonstrated in Table 2. Out of the 32 cases, there were 15 patients with definite follow-ups. A total of nine patients with lymph node metastasis died due to recurrence, of which six cases showed positive expression of both PIK3CA and survivin, and three cases showed positive expression of either PIK3CA or survivin. Furthermore, the four cases that died within 1 year of the surgical resection were all positive for PIK3CA and survivin expression. Two cases achieved stable disease (SD) after the surgery, and both displayed negative expression of PIK3CA and survivin (Table 3). The relationships between PIK3CA and survivin expression The relationship between PIK3CA and survivin expression was studied among 32 ASC patients. Our results showed that ASC could be classified into four groups. In group 1, PIK3CA and survivin demonstrated positive staining. In
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The incidence of adenosquamous lung carcinoma varies from 0.4 to 4 % of all lung cancers according to the WHO Classification of Tumors, which was published in 2004 [1]. This low incidence of ASC might be attributed to three main factors. First, the preoperative biopsy specimen often leads to removal of the AC or only the SCC component, which cannot be easily found. Secondly, it is difficult to histologically classify the ASC because of the undifferentiated cells. Finally, there are no studies with adequate size of patient population for a definitive statistical analysis of the nature of ASC. Heterogeneity is a kind of character, which frequently occurs in cancer. ASC is an atypical heterogeneous tumor including two distinct components of AC and SC. When it comes to the histogenesis of ASC, there are many hypotheses, including AC with squamous metaplasia, collision tumor, high-grade mucoepidermoid carcinoma, and bipotential undifferentiated cell origin [7]. Niho and colleagues [8] reported that SC and AC components showed identical monoclonal patterns, which suggested that both of these components originated from the same cell type. Kanazawa et al. [9] also suggested the monoclonal transition from SC to AC in the carcinogenic process. The PI3K activation is commonly observed in many human cancer cells. While survivin expression is elevated in cancer cells, it is further induced by some growth factors through PI3K activation. The PI3K/Akt signaling pathway has been implicated in the up-regulation of survivin in both vascular endothelial and tumor cells [10]. Consequently, in our study, we explored whether PIK3CA and survivin
Med Oncol (2014) 31:983
expressions were up-regulated in ASC. Many studies mentioned that a high lymph node metastasis might lead to an adverse prognosis. Filosso and colleagues reported that an aggressive biological behavior of ASC was demonstrated by a high lymph node metastases percentage at presentation (29 patients, 60.4 %), which could translate into a higher pathological stage at the intervention (ASC stages II, III, and IV: 33/48, p = 0.0001) [11]. Ichiro and colleagues reported that in comparison with the negative staining group, the presence of node metastasis was significantly higher in the group that stained positive for PIK3CA in esophageal squamous cell carcinoma [12]. Li et al. [13] pointed out that a parallel molecular detection of survivin and livin mRNAs was a specific diagnostic tool for the assessment of lymph node micrometastasis, which could show powerful prognostic implications for the patients with a completely resected stage I NSCLC. As for our study, the outcome revealed that the positive rate of PIK3CA and survivin expressions in ASC had a remarkable relationship with the lymph node metastasis, which was in line with the above-mentioned reports. The range of the lymph node metastasis was a vital prognostic factor for the patients. As the lung ASC has an infrequent occurrence and is very complex, an early diagnosis of the lymph node metastasis is difficult but necessary. Taken this into the consideration, a high expression of PIK3CA and survivin in the adenosquamous lung carcinoma might act as a predictor for lymph node metastasis. The biological behavior and clinicopathological characteristics of ASC have not yet been thoroughly unraveled. Among the 15 patients with follow-ups, there were 9 patients with lymph node metastasis (either PIK3CA- or survivin-positive expression) that died due to recurrence, such as brain and bone distant metastasis. Furthermore, four cases died within 1 year of the surgical resection, and all of them were positive for PIK3CA and survivin expression. These results suggested that positive expression of PIK3CA and survivin was associated with adverse prognosis, especially for double positivity. Alternatively, double-negative expression of PIK3CA and survivin might suggest a good prognosis. The similar results were also observed in Lin’s study that demonstrated an overexpression of PI3K might be linked to the process of tumor-cell transformation in lung carcinomas, including regional lymph nodes metastasis and the occurrence of distant metastases [14]. In the series by Guo and colleagues, they found that the expression of survivin and p-Akt was positively correlated to the tumor recurrence in the HCC patients. With regard to our study, there might be a potential tendency toward the relationship between the expression of PIK3CA and survivin with recurrence. However, there were still some weak points in this paper. To our knowledge, IHC staining has often been used
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for semi-quantitative measurement of the proteins expressed in the tissues. However, this method might be limited by variation according to the different antibodies used. Therefore, other methods should further support the results in the present study, such as real-time PCR and Western blot. Moreover, more patients and follow-ups will be required. In summary, we suggested that the expression of PIK3CA and survivin had a significant correlation with the TNM stage, pathological grade, and lymph node metastasis. Further clinical studies should be carried out on the patients with adenosquamous lung carcinoma in order to determine the relationships between the PIK3CA- and survivin-positive expression and recurrence. PIK3CA and survivin might be suggested as potential targets for the development of therapeutic strategies for adenosquamous lung carcinoma. Acknowledgments This work was supported by grants from the National Nature Science Foundation of China (81071228), Medical Important Talents of Jiangsu Province (RC201157), and Project of Oncology Translational Medicine Central of Jiangsu Province (BL2012008). Conflict of interest financial one.
There was no conflict of interest including any
References 1. Travis W, Brambilla E, Muller-Hermelink H, Harris C. Tumours of the lung, pleura, thymus and heart. Pathology and genetics. World Health Organization Classification of Tumours. Lyon: IARC Press; 2004. 2. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene. 2008;27(41):5497–510. doi:10. 1038/onc.2008.245. 3. De Luca A, Maiello MR, D’Alessio A, Pergameno M, Normanno N. The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets. 2012;16(S2): S17–27. 4. Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res. 2008;14(16):5000–5. 5. Yamamoto T, Tanigawa N. The role of survivin as a new target of diagnosis and treatment in human cancer. Med Electron Microsc. 2001;34(4):207–12. 6. Goldstraw P. Site-specific explanatory notes for lung and pleural tumours. International Association for the Study of Lung Cancer Staging Handbook in Thoracic Oncology. Orange Park: Editorial Rx Press; 2009. p. 53–74. 7. Ichinose Y, Hara N, Takamori S, Maeda K, Yano T, Ohta M. DNA ploidy pattern of each carcinomatous component in adenosquamous lung carcinoma. Ann Thorac Surg. 1993;55(3): 593–6. 8. Niho S, Yokose T, Kodama T, Nishiwaki Y, Mukai K. Clonal analysis of adenosquamous carcinoma of the lung. Cancer Sci. 1999;90(11):1244–7. 9. Kanazawa H, Ebina M, Ino-oka N, Shimizukawa M, Takahashi T, Fujimura S, et al. Transition from squamous cell carcinoma to
123
983 Page 6 of 6 adenocarcinoma in adenosquamous carcinoma of the lung. Am J Pathol. 2000;156(4):1289–98. 10. Zhao P, Meng Q, Liu LZ, You YP, Liu N, Jiang BH. Regulation of survivin by PI3K/Akt/p70S6K1 pathway. Biochem Biophys Res Commun. 2010;395(2):219–24. doi:10.1016/j.bbrc.2010.03. 165. 11. Maeda H, Matsumura A, Kawabata T, Suito T, Kawashima O, Watanabe T, et al. Adenosquamous carcinoma of the lung: surgical results as compared with squamous cell and adenocarcinoma cases. Eur J Cardio-thorac Surg Off J Eur Assoc Cardiothorac Surg. 2012;41(2):357–61. doi:10.1016/j.ejcts.2011.05.050. 12. Akagi I, Miyashita M, Makino H, Nomura T, Hagiwara N, Takahashi K, et al. Overexpression of PIK3CA is associated with
123
Med Oncol (2014) 31:983 lymph node metastasis in esophageal squamous cell carcinoma. Int J Oncol. 2009;34(3):767. 13. Li J, Li ZN, Yu LC, Shi SB, Ge LP, Wu JR, et al. Gene diagnosis of micrometastases in regional lymph nodes of patients with stage I non-small cell lung cancer: impact on staging and prognosis. Clin Transl Oncol Off Publ Fed Span Oncol Soc Natl Cancer Inst Mexico. 2013;. doi:10.1007/s12094-013-1017-1. 14. Lin X, Bohle AS, Dohrmann P, Leuschner I, Schulz A, Kremer B, et al. Overexpression of phosphatidylinositol 3-kinase in human lung cancer. Langenbeck’s Arch Surg. 2001;386(4):293–301.
