Ann Surg Oncol DOI 10.1245/s10434-015-4576-2
ORIGINAL ARTICLE – THORACIC ONCOLOGY
Bis Expression in Patients with Surgically Resected Lung Cancer and its Clinical Significance Chang Dong Yeo, MD, PhD1, Gyeong Sin Park, MD, PhD2,3, Nahyeon Kang, MSc1,3, Su Yeon Choi, BSc1,3, Hye Yun Kim, MSc3,4,5, Dong Soo Lee, MD, PhD6, Yeon Sil Kim, MD, PhD6, Young Kyoon Kim, MD, PhD1, Jae Gil Park, PhD7, Sook Whan Sung, MD, PhD7, Kyo Young Lee, MD, PhD2, Mi Sun Park, MSc8,9, Hyeon Woo Yim, MD, PhD8,9, Seung Joon Kim, MD, PhD1,3, and Jeong-Hwa Lee, MD, PhD3,4,5 1
Division of Pulmonology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; 2Department of Hospital Pathology, The Catholic University of Korea, Seoul, Republic of Korea; 3The Cancer Research Institute, The Catholic University of Korea, Seoul, Republic of Korea; 4Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; 5Cancer Evolution Research Center, The Catholic University of Korea, Seoul, Republic of Korea; 6Department of Radiation Oncology, The Catholic University of Korea, Seoul, Republic of Korea; 7Department of Thoracic Surgery, The Catholic University of Korea, Seoul, Republic of Korea; 8Department of Biostatistics, The Catholic University of Korea, Seoul, Republic of Korea; 9Clinical Research Coordinating Center, The Catholic University of Korea, Seoul, Republic of Korea
ABSTRACT Background. Bis, also known as BAG3, has been identified as a Bcl-2-interacting protein that enhances cellular anti-apoptotic activity. It is involved in cellular differentiation, angiogenesis, migration, and invasion in various tumors. The purpose of this study was to investigate the Bis expression pattern, and the clinical significance thereof, in patients with resected lung cancer. Methods. We studied 121 lung cancer patients who underwent curative surgical resection. Patient clinicopathological characteristics were reviewed retrospectively from medical records, including tumor recurrence and survival. The expression of Bis protein in lung cancer tissues was evaluated by immunohistochemical staining and was assessed using a four-
Chang Dong Yeo and Gyeong Sin Park have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-015-4576-2) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2015 First Received: 15 March 2015 S. J. Kim, MD, PhD e-mail: [email protected] J.-H. Lee, MD, PhD e-mail: [email protected]
tiered intensity score system (negative, weak, moderate, strong). Enhanced Bis expression at the periphery of a tumor facing the adjacent nontumor region was referred as ‘‘marginal activity.’’ Results. Although Bis expression was higher in squamous cell carcinoma than in adenocarcinoma, marginal activity was higher in adenocarcinoma than in squamous cell carcinoma. All of the small cell carcinomas and lung cancer with neuroendocrine differentiation examined were negative for Bis expression. Compared with stage I lung cancer, patients with stage II and IIIA lung cancer exhibited higher Bis protein levels in lung tissues. Recurrence and survival rates did not differ significantly according to Bis expression intensity score or marginal activity. Conclusions. Our study demonstrated that Bis expression differed according to the histological type and pathological stage of the lung cancer. Further studies are needed to assess its use as a biomarker and its role in the molecular pathogenesis of lung cancer.
Lung cancer remains the leading cause of cancer death worldwide. In 2008, patients receiving a new diagnosis of lung cancer represented 13 % of all new cancer diagnoses, and 1.4 million died of lung cancer, 18 % of all cancer deaths.1 For patients with early-stage non-small-cell lung cancer (NSCLC), surgical resection is the treatment of choice for curative intent. The prognoses for stage I and II
C. D. Yeo et al.
NSCLC, expressed in terms of 5-year survival rates, are commonly accepted to be 60–80 % for stage I and 30–50 % for stage II NSCLC.2,3 Tumor recurrence, including local recurrence and distant metastasis, is the main obstacle for long-term survival after resection.4 Thus, several biomarkers in surgical specimens have been analyzed as prognostic and predictive for recurrence after complete resection.5–7 Bis (Bcl-2-interacting cell death suppressor), also known as BAG3 and CAIR-1, has been identified as a Bcl2–interacting protein that enhances cellular anti-apoptotic activity.8,9 Accumulating evidence indicates that Bis also is involved in cellular processes, including protein homeostasis, cellular differentiation, angiogenesis, migration, and invasion, through its interaction with various binding partners.10,11 Several studies have demonstrated that downregulation of Bis increased the sensitivity of tumor cells against various stimuli, such as proteasome inhibitors, oxidative stress, and chemotherapeutic agents.12–14 The prosurvival activity of Bis was further confirmed by in vivo studies showing constitutive high-level expression of Bis in various tumors of different origins, including neoplastic leukocytes, pancreatic cancer, thyroid cancer, prostate cancer, and chronic lymphocytic leukemia.12,15–18 Additionally, higher expression of Bis shows a positive correlation with poor survival and more aggressive cancers.19,20 These results raise the possibility that Bis could be potentially useful as a biomarker or even a therapeutic target for various human malignant neoplasms. Previously, Bis protein was found to be highly expressed in 27.3 % (6/22 cases) of NSCLC and silencing of Bis induced apoptosis in vitro.21 In small-cell lung cancer (SCLC), downregulation of Bis increased cell death and sensitized cells to cisplatin treatment.22 However, little is known about the clinical significance of Bis expression in NSCLC. Thus, in this study, we investigated the Bis
expression profile, and the clinical significance thereof, in 121 cases of lung cancer. METHODS We selected 121 consecutive NSCLC and SCLC patients who gave written, informed consent for use of their clinical material and underwent curative surgical resection of a primary lesion at Seoul St. Mary’s Hospital. Patient clinicopathological characteristics were reviewed retrospectively from medical records, including demographic TABLE 1 Characteristics of 107 patients with surgically resected adenocarcinoma and squamous cell carcinoma Variable
Value (n = 107)
Gender Male
74 (69.2 %)
Female
33 (30.8 %)
Age (years)
64.6 ± 8.2
Smoking status Never smoker
35 (32.7 %)
Ex-smoker Current smoker
55 (51.4 %) 15 (14.0 %)
Unknown
2 (1.9 %)
Pathology Adenocarcinoma
57 (53.3 %)
Squamous cell carcinoma
50 (46.7 %)
T stage T1
33 (30.8 %)
T2
59 (55.1 %)
T3
14 (13.1 %)
T4
1 (0.9 %)
N stage N0
58 (54.2 %)
N1
24 (22.4 %)
N2
25 (23.4 %)
Stage I
42 (39.3 %)
II
34 (31.8 %)
IIIA
31 (29.0 %)
Bis expression intensity score Negative
10 (0–50)
Weak
20 (0–30)
Moderate
30 (10–50)
Strong
10 (0–40)
Marginal activity
FIG. 1 Marginal Bis expression; strong expression in the marginal (left) but negative in the central (right) area. 940 (inset 9400)
Center, margin: equal
83 (77.6 %)
Center: negative, margin: positive
13 (12.1 %)
Center: weak, margin: strong
11 (10.3 %)
Values are numbers (percentages) for categorical variables, and means ± SD or median (IQR) for continuous variables
Bis Expression in Patients with Surgically Resected Lung Cancer and its Clinical Significance TABLE 2 Bis expression according to the histology Variable
Adenocarcinoma (n = 57)
Squamous cell carcinoma (n = 50)
P value
Expression intensity score Negative
30 (0–60)
0 (0–10)
0.0002
Positive (weak ? moderate ? strong)
70 (40–100)
100 (90–100)
0.0002
Center, margin: equal
37 (64.9 %)
46 (92.0 %)
0.0023
Center: negative, margin: positive
12 (21.1 %)
1 (2.0 %)
8 (14.0 %)
3 (6.0 %)
Marginal activity
Center: weak, margin: strong
Values are numbers (percentages) for categorical variables, and medians (IQR) for continuous variables
FIG. 2 Bis expression in various cell types in lung cancer. a Smallcell carcinoma: negative; b poorly differentiated carcinoma with neuroendocrine differentiation: negative; c large cell neuroendocrine carcinoma: negative; d mixed squamous cell carcinoma and small cell carcinoma: negative in small cell carcinoma component (asterisk) and
strong in squamous cell carcinoma component; e adenosquamous cell carcinoma: negative in adenocarcinoma component and weak in squamous cell carcinoma component (open star); f large-cell carcinoma: strong
data, histological type, and pathological (TNM) stage. Pathological staging was reclassified on the basis of the most recent (seventh) edition of the International Association for the Study of Lung Cancer Staging Project.23 The study protocol was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea (Approval No. KC14SISI0059). Postoperative adjuvant chemotherapy with platinumbased regimens was given to patients with stage II or IIIA as a standard, and follow-up examination consisted of routine medical check-ups, including chest computed tomography at 6-month intervals for the initial 5 years and then annually thereafter. Tumor recurrence was diagnosed using radiological examinations. If possible, recurrence was confirmed cytologically and/or pathologically. The day of first detection of any recurrence was defined as the date of diagnosis of recurrence.
Formalin-fixed, paraffin wax-embedded tissue blocks of lung cancer tissue were obtained from surgical specimens. The expression of Bis protein in lung cancer tissues was evaluated by immunohistochemical staining performed on 4-lm, paraffin-wax-embedded tissue sections. Briefly, paraffin wax sections were mounted on coated slides and deparaffinized, and then rehydrated through an ethanol series. The slides were subjected to antigen retrieval with the PTLink microwave system (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with 0.3 % hydrogen peroxide for 5 min. The sections were incubated with an anti-Bis antibody (dilution 1:5000) for 30 min at room temperature, as described.8 After washing, a goat antirabbit-HRP secondary antibody (Biocare, Concord, CA) was applied and incubated for a further 20 min. The slides were visualized with Betazoid DAB Chromogen Kit (Biocare), followed by hematoxylin counterstaining.
C. D. Yeo et al. TABLE 3 Bis expression and pathological stages in patients with adenocarcinoma and squamous cell carcinoma Variable
Stage I (n = 42)
Stage II (n = 34)
Stage IIIA (n = 31)
P value
Expression intensity score Negative
30 (10–60)
0 (0–20)
10 (0–30)
0.001*
Positive (weak ? moderate ? strong)
70 (40–90)
100 (80–100)
90 (70–100)
0.001* 0.6218
Marginal activity Center, margin: equal
32 (76.2 %)
28 (82.4 %)
23 (74.2 %)
Center: negative, margin: positive
7 (16.7 %)
2 (5.9 %)
4 (12.9 %)
Center: weak, margin: strong
3 (7.1 %)
4 (11.8 %)
4 (12.9 %)
Values are numbers (percentages) for categorical variables, and medians (IQR) for continuous variables * Significant differences between stage I versus stage II ? stage IIIA, by multiple comparisons from Kruskal–Wallis test, followed by a post hoc Dunn’s test
Bis expression was assessed by two independent pathologists (GSP and GYL) using a four-tiered intensity score system (negative, weak, moderate, strong) according to the expression in adjacent nontumor tissues (Suppl. Fig. 1). Supplemental Fig. 2 shows the Bis expression in resected lung cancer tissues (adenocarcinoma and squamous cell carcinoma). Additionally, scores were expressed as percentages. Enhanced Bis expression at the periphery of a tumor facing the adjacent nontumor region was referred to as ‘‘marginal activity,’’ which was compared with the expression at the tumor center (Fig. 1). Specimens that were scored differently by the two pathologists were reexamined together to reach agreement. Statistical Analysis Demographic data are presented as numbers (%), mean ± SDs, or medians (IQR). A comparison of categorical variables was made using the v2 test or Fisher’s exact test. For continuous variables, the Wilcoxon rank-sum test was used for two groups and the Kruskal–Wallis test (with Dunn’s multiple comparison test) for more than two groups. The prognostic significance of factors affecting overall survival was determined using the Cox proportional hazard ratio (HR) model, whereas factors affecting the cumulative incidence of recurrence were determined using the semiparametric proportional hazards model for subdistribution of competing risk analysis (SAS macro ‘‘%PSHREG’’). A twosided P value \0.05 was considered to indicate statistical significance. All statistical analyses were performed using the SAS software (ver. 9.3; SAS Institute, Inc., Cary, NC). RESULTS Among the total 121 lung cancer patients, 115 were NSCLC (57 adenocarcinomas, 50 squamous cell carcinomas, 3 adenosquamous cell carcinoma, 1 poorly differentiated carcinoma with neuroendocrine differentiation, 1 large cell
neuroendocrine carcinoma, 1 mixed squamous cell carcinoma and small cell carcinoma, 1 large cell carcinoma, and 1 double primary tumor with adenocarcinoma plus squamous cell carcinoma) patients from February 2009 to December 2013, and 6 were SCLC patients from January 2002 to January 2014. Statistical analyses were performed on 107 NSCLC patients with adenocarcinomas and squamous cell carcinomas (Table 1). The mean age was 64.6 years; there were 74 (69.2 %) males and 33 (30.8 %) females. Pathological stages I, II, and IIIA were 39.3, 31.8, and 29.0 %, respectively. Table 2 shows the differences in Bis expression between adenocarcinoma and squamous cell carcinoma. Patients with squamous cell carcinomas had higher Bis expression than those with adenocarcinomas. However, marginal activity was higher in patients with adenocarcinomas than in those with squamous cell carcinomas. All six SCLC cases, one poorly differentiated carcinoma with neuroendocrine differentiation, one large cell neuroendocrine carcinoma, and one small cell carcinoma component of mixed squamous cell carcinoma and small cell carcinoma were negative for Bis expression. However, one large cell carcinoma showed strong expression of Bis protein in lung tissues (Fig. 2). Compared with stage I lung cancer, patients with stage II and IIIA lung cancer expressed higher Bis protein levels in their lung tissues (P = 0.001; Table 3). Compared with stage I adenocarcinoma, patients with stage II and IIIA adenocarcinoma expressed higher Bis protein levels (P = 0.0135; Suppl. Table 1). Among the 107 resected lung cancer patients, 38 (35.5 %) patients showed recurrence and 28 (26.2 %) died. The median relapse-free duration was 13.3 months, and median overall survival was 32.2 months. Relapse-free duration and overall survival did not differ significantly between adenocarcinomas and squamous cell carcinomas (data not shown). Moreover, recurrence and survival rate did not differ significantly according to Bis expression intensity scores or marginal activity (Suppl. Tables 2, 3).
Bis Expression in Patients with Surgically Resected Lung Cancer and its Clinical Significance
DISCUSSION The purpose of the present study was to investigate Bis expression patterns in patients with resected lung cancer and to assess its prognostic value. We observed that patients with squamous cell carcinomas had higher Bis expression than those with adenocarcinomas. However, patients with adenocarcinomas had higher marginal activity than those with squamous cell carcinomas. Bis protein was not expressed in SCLC, small cell carcinoma components of NSCLC, or NSCLC with neuroendocrine features. Higher Bis expression was also noted in patients with stage II and IIIA NSCLC than those with stage I. However, there was no significant difference in recurrence or overall survival according to Bis expression intensity scores or marginal activity. Bis is expressed ubiquitously in various tissues and is involved in the regulation of biological processes, such as apoptosis, development, cytoskeleton organization, and autophagy.24 Bis is a stress- and survival-related protein that has been shown to be upregulated in cellular stress, such as exposure to high temperature, heavy metals, proteasome inhibitors, and oxidative stress.11,14,25,26 In addition to stressed cells, in vitro, Bis is upregulated in various types of cancer and underlies resistance to therapy, through downmodulation of apoptosis.12 However, few studies related to lung cancer have been reported. Zhang et al. demonstrated that Bis and Hsp70 protein were expressed in the cytoplasm in 27.3 % (6/22) of cases of NSCLC, whereas weak Hsp70 and no Bis expression were detected in human normal bronchial epithelial cells.21 Moreover, the silencing of Bis resulted in diminished levels of Bcl-2 protein. However, the study did not evaluate the clinical characteristics of the lung cancer patients with Bis expression in lung cancer tissues or its prognostic role in recurrence or survival. Bis expression patterns in different lung cancer tissues were demonstrated using immunohistochemistry by Chiappetta et al.22 They showed Bis expression in squamous cell carcinoma was higher than that in adenocarcinoma, consistent with our results. However, they reported that 38.9 % (14/36) of SCLC cases showed no Bis expression and no Bis expression was detected in normal lung. In the present study, Bis protein was not expressed in any of the six SCLCs tested, one small cell carcinoma component of NSCLC, or two NSCLC with neuroendocrine features. However, Bis expression was detected in normal lung tissue, including type II pneumocytes, bronchial epithelial reserve cells, endothelial cells, and smooth muscle cells. One possible explanation for the discrepancy between the studies is the relatively small number of SCLC cases in our study. Additionally, we analyzed surgically resected lung tissues with pathological staging, whereas the previous study did not describe the tissue source: whether it was small tissue samples from bronchoscopic or needle
biopsies, or surgically resected tissue samples. The major concern of our study was to investigate Bis expression with clinical findings, whereas the previous study focused on the anti-apoptotic effect of Bis in SCLC. To our knowledge, this is the first report of Bis expression in lung cancer tissues according to histological classification and clinical significance. In the current study, although Bis expression was higher in squamous cell types, recurrence and survival did not differ between squamous cell carcinoma and adenocarcinoma. It has been reported that the squamous cell type of NSCLC is closely related to smoking status.27 We hypothesized that environmental stresses, such as smoking and oxidants, were more likely to promote expression of Bis protein, which is supported by high Hsp70 expression in smokers and patients with chronic obstructive pulmonary disease.28 In epithelial cell lines, a high level of Bis expression was associated with cell adhesion and migration, supporting a role for Bis in tumor invasion and metastasis.29,30 Furthermore, downmodulation of Bis decreased invasive ability in glioma cells.31 These findings are compatible with our data, showing that Bis expression was higher in patients with stage II and IIIA than those with stage I disease. Additionally, the marginal activity of Bis was significantly higher than in the center, which was more prominent in adenocarcinomas, suggesting a positive role of Bis in the motility of lung cancer cells. Regarding the molecular mechanism through which Bis modulates invasion and motility, it has been noted that Bis can interact with various signaling proteins, including CCT, guanine nucleotide exchange factor 2, and MMP-2, through the PXXP or WW domains of the Bis protein.30,32–34 However, the critical involvement of Bis in the metastasis of lung cancer should be studied using tissues from metastatic regions as well as primary sites in future studies. Our study had several limitations. First, the number of lung cancer patients was insufficient to make conclusions of clinical significance. Second, we did not measure the expression of Bcl-2, Hsp70, or Bax, important apoptotic proteins involved in the pathogenesis of Bis expression. Third, we did not consider the effect of adjuvant chemotherapy in this analysis. Cisplatin-based adjuvant chemotherapy improves survival among patients with completely resected stage II and IIIA NSCLC.35 Additionally, Bis upregulation could influence chemotherapyinduced apoptosis in neoplastic cells.26
CONCLUSIONS Our study demonstrated that Bis expression in patients with resected lung cancer differed according to histological
C. D. Yeo et al.
type and pathological stage. Further studies are needed to assess its value as a biomarker for prognosis in various types of lung cancer. ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) Grants, funded by the Korean Government (MSIP) (Nos. 2014R1A2A1A11052422, 2012R1A1A2 007589 and 2012R1A5A2047939). DISCLOSURES interests.
The authors declare that they have no competing
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16. Chiappetta G, Ammirante M, Basile A, et al. The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosisinducing ligand. J Clin Endocrinol Metab. 2007;92:1159–63. 17. Staibano S, Mascolo M, Di Benedetto M, et al. BAG3 protein delocalisation in prostate carcinoma. Tumour Biol. 2010;31:461–9. 18. Zhu H, Wu W, Fu Y, et al. Overexpressed BAG3 is a potential therapeutic target in chronic lymphocytic leukemia. Ann Hematol. 2014;93:425–35. 19. Festa M, Del Valle L, Khalili K, et al. BAG3 protein is overexpressed in human glioblastoma and is a potential target for therapy. Am J Pathol. 2011;178:2504–12. 20. Rosati A, Bersani S, Tavano F, et al. Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival. Am J Pathol. 2012;181:1524–9. 21. Zhang Y, Wang JH, Lu Q, Wang YJ. Bag3 promotes resistance to apoptosis through Bcl-2 family members in non-small cell lung cancer. Oncol Rep. 2012;27:109–13. 22. Chiappetta G, Basile A, Barbieri A, et al. The anti-apoptotic BAG3 protein is expressed in lung carcinomas and regulates small cell lung carcinoma (SCLC) tumor growth. Oncotarget. 2014;5(16):6846–53. 23. Goldstraw P, Crowley J, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol. 2007;2:706–14. 24. Rosati A, Graziano V, De Laurenzi V, et al. BAG3: a multifaceted protein that regulates major cell pathways. Cell Death Dis. 2011;2:e141. 25. Wang HQ, Liu HM, Zhang HY, et al. Transcriptional upregulation of BAG3 upon proteasome inhibition. Biochem Biophys Res Commun. 2008;365:381–5. 26. Zhu H, Liu P, Li J. BAG3: a new therapeutic target of human cancers? Histol Histopathol. 2012;27:257–61. 27. de Groot P, Munden RF. Lung cancer epidemiology, risk factors, and prevention. Radiol Clin North Am. 2012;50:863–76. 28. Dong J, Guo L, Liao Z, et al. Increased expression of heat shock protein 70 in chronic obstructive pulmonary disease. Int Immunopharmacol. 2013;17:885–93. 29. Iwasaki M, Homma S, Hishiya A, et al. BAG3 regulates motility and adhesion of epithelial cancer cells. Cancer Res. 2007;67:10252–9. 30. Suzuki M, Iwasaki M, Sugio A, et al. BAG3 (BCL2-associated athanogene 3) interacts with MMP-2 to positively regulate invasion by ovarian carcinoma cells. Cancer Lett. 2011;303:65–71. 31. Lee YD, Cui MN, Yoon HH, et al.: Down-modulation of Bis reduces the invasive ability of glioma cells induced by TPA, through NF-kappaB mediated activation of MMP-9. BMB Rep. 2014;47:262–7. 32. Kassis JN, Guancial EA, Doong H, et al. CAIR-1/BAG-3 modulates cell adhesion and migration by downregulating activity of focal adhesion proteins. Exp Cell Res. 2006;312:2962–71. 33. Fontanella B, Birolo L, Infusini G, et al. The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: new hints for actin folding. Int J Biochem Cell Biol. 2010;42:641–50. 34. Iwasaki M, Tanaka R, Hishiya A, et al. BAG3 directly associates with guanine nucleotide exchange factor of Rap1, PDZGEF2, and regulates cell adhesion. Biochem Biophys Res Commun. 2010;400:413–8. 35. Pignon JP, Tribodet H, Scagliotti GV, et al. Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group. J Clin Oncol. 2008;26:3552–9.
ORIGINAL ARTICLE – THORACIC ONCOLOGY
Bis Expression in Patients with Surgically Resected Lung Cancer and its Clinical Significance Chang Dong Yeo, MD, PhD1, Gyeong Sin Park, MD, PhD2,3, Nahyeon Kang, MSc1,3, Su Yeon Choi, BSc1,3, Hye Yun Kim, MSc3,4,5, Dong Soo Lee, MD, PhD6, Yeon Sil Kim, MD, PhD6, Young Kyoon Kim, MD, PhD1, Jae Gil Park, PhD7, Sook Whan Sung, MD, PhD7, Kyo Young Lee, MD, PhD2, Mi Sun Park, MSc8,9, Hyeon Woo Yim, MD, PhD8,9, Seung Joon Kim, MD, PhD1,3, and Jeong-Hwa Lee, MD, PhD3,4,5 1
Division of Pulmonology, Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; 2Department of Hospital Pathology, The Catholic University of Korea, Seoul, Republic of Korea; 3The Cancer Research Institute, The Catholic University of Korea, Seoul, Republic of Korea; 4Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; 5Cancer Evolution Research Center, The Catholic University of Korea, Seoul, Republic of Korea; 6Department of Radiation Oncology, The Catholic University of Korea, Seoul, Republic of Korea; 7Department of Thoracic Surgery, The Catholic University of Korea, Seoul, Republic of Korea; 8Department of Biostatistics, The Catholic University of Korea, Seoul, Republic of Korea; 9Clinical Research Coordinating Center, The Catholic University of Korea, Seoul, Republic of Korea
ABSTRACT Background. Bis, also known as BAG3, has been identified as a Bcl-2-interacting protein that enhances cellular anti-apoptotic activity. It is involved in cellular differentiation, angiogenesis, migration, and invasion in various tumors. The purpose of this study was to investigate the Bis expression pattern, and the clinical significance thereof, in patients with resected lung cancer. Methods. We studied 121 lung cancer patients who underwent curative surgical resection. Patient clinicopathological characteristics were reviewed retrospectively from medical records, including tumor recurrence and survival. The expression of Bis protein in lung cancer tissues was evaluated by immunohistochemical staining and was assessed using a four-
Chang Dong Yeo and Gyeong Sin Park have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-015-4576-2) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2015 First Received: 15 March 2015 S. J. Kim, MD, PhD e-mail: [email protected] J.-H. Lee, MD, PhD e-mail: [email protected]
tiered intensity score system (negative, weak, moderate, strong). Enhanced Bis expression at the periphery of a tumor facing the adjacent nontumor region was referred as ‘‘marginal activity.’’ Results. Although Bis expression was higher in squamous cell carcinoma than in adenocarcinoma, marginal activity was higher in adenocarcinoma than in squamous cell carcinoma. All of the small cell carcinomas and lung cancer with neuroendocrine differentiation examined were negative for Bis expression. Compared with stage I lung cancer, patients with stage II and IIIA lung cancer exhibited higher Bis protein levels in lung tissues. Recurrence and survival rates did not differ significantly according to Bis expression intensity score or marginal activity. Conclusions. Our study demonstrated that Bis expression differed according to the histological type and pathological stage of the lung cancer. Further studies are needed to assess its use as a biomarker and its role in the molecular pathogenesis of lung cancer.
Lung cancer remains the leading cause of cancer death worldwide. In 2008, patients receiving a new diagnosis of lung cancer represented 13 % of all new cancer diagnoses, and 1.4 million died of lung cancer, 18 % of all cancer deaths.1 For patients with early-stage non-small-cell lung cancer (NSCLC), surgical resection is the treatment of choice for curative intent. The prognoses for stage I and II
C. D. Yeo et al.
NSCLC, expressed in terms of 5-year survival rates, are commonly accepted to be 60–80 % for stage I and 30–50 % for stage II NSCLC.2,3 Tumor recurrence, including local recurrence and distant metastasis, is the main obstacle for long-term survival after resection.4 Thus, several biomarkers in surgical specimens have been analyzed as prognostic and predictive for recurrence after complete resection.5–7 Bis (Bcl-2-interacting cell death suppressor), also known as BAG3 and CAIR-1, has been identified as a Bcl2–interacting protein that enhances cellular anti-apoptotic activity.8,9 Accumulating evidence indicates that Bis also is involved in cellular processes, including protein homeostasis, cellular differentiation, angiogenesis, migration, and invasion, through its interaction with various binding partners.10,11 Several studies have demonstrated that downregulation of Bis increased the sensitivity of tumor cells against various stimuli, such as proteasome inhibitors, oxidative stress, and chemotherapeutic agents.12–14 The prosurvival activity of Bis was further confirmed by in vivo studies showing constitutive high-level expression of Bis in various tumors of different origins, including neoplastic leukocytes, pancreatic cancer, thyroid cancer, prostate cancer, and chronic lymphocytic leukemia.12,15–18 Additionally, higher expression of Bis shows a positive correlation with poor survival and more aggressive cancers.19,20 These results raise the possibility that Bis could be potentially useful as a biomarker or even a therapeutic target for various human malignant neoplasms. Previously, Bis protein was found to be highly expressed in 27.3 % (6/22 cases) of NSCLC and silencing of Bis induced apoptosis in vitro.21 In small-cell lung cancer (SCLC), downregulation of Bis increased cell death and sensitized cells to cisplatin treatment.22 However, little is known about the clinical significance of Bis expression in NSCLC. Thus, in this study, we investigated the Bis
expression profile, and the clinical significance thereof, in 121 cases of lung cancer. METHODS We selected 121 consecutive NSCLC and SCLC patients who gave written, informed consent for use of their clinical material and underwent curative surgical resection of a primary lesion at Seoul St. Mary’s Hospital. Patient clinicopathological characteristics were reviewed retrospectively from medical records, including demographic TABLE 1 Characteristics of 107 patients with surgically resected adenocarcinoma and squamous cell carcinoma Variable
Value (n = 107)
Gender Male
74 (69.2 %)
Female
33 (30.8 %)
Age (years)
64.6 ± 8.2
Smoking status Never smoker
35 (32.7 %)
Ex-smoker Current smoker
55 (51.4 %) 15 (14.0 %)
Unknown
2 (1.9 %)
Pathology Adenocarcinoma
57 (53.3 %)
Squamous cell carcinoma
50 (46.7 %)
T stage T1
33 (30.8 %)
T2
59 (55.1 %)
T3
14 (13.1 %)
T4
1 (0.9 %)
N stage N0
58 (54.2 %)
N1
24 (22.4 %)
N2
25 (23.4 %)
Stage I
42 (39.3 %)
II
34 (31.8 %)
IIIA
31 (29.0 %)
Bis expression intensity score Negative
10 (0–50)
Weak
20 (0–30)
Moderate
30 (10–50)
Strong
10 (0–40)
Marginal activity
FIG. 1 Marginal Bis expression; strong expression in the marginal (left) but negative in the central (right) area. 940 (inset 9400)
Center, margin: equal
83 (77.6 %)
Center: negative, margin: positive
13 (12.1 %)
Center: weak, margin: strong
11 (10.3 %)
Values are numbers (percentages) for categorical variables, and means ± SD or median (IQR) for continuous variables
Bis Expression in Patients with Surgically Resected Lung Cancer and its Clinical Significance TABLE 2 Bis expression according to the histology Variable
Adenocarcinoma (n = 57)
Squamous cell carcinoma (n = 50)
P value
Expression intensity score Negative
30 (0–60)
0 (0–10)
0.0002
Positive (weak ? moderate ? strong)
70 (40–100)
100 (90–100)
0.0002
Center, margin: equal
37 (64.9 %)
46 (92.0 %)
0.0023
Center: negative, margin: positive
12 (21.1 %)
1 (2.0 %)
8 (14.0 %)
3 (6.0 %)
Marginal activity
Center: weak, margin: strong
Values are numbers (percentages) for categorical variables, and medians (IQR) for continuous variables
FIG. 2 Bis expression in various cell types in lung cancer. a Smallcell carcinoma: negative; b poorly differentiated carcinoma with neuroendocrine differentiation: negative; c large cell neuroendocrine carcinoma: negative; d mixed squamous cell carcinoma and small cell carcinoma: negative in small cell carcinoma component (asterisk) and
strong in squamous cell carcinoma component; e adenosquamous cell carcinoma: negative in adenocarcinoma component and weak in squamous cell carcinoma component (open star); f large-cell carcinoma: strong
data, histological type, and pathological (TNM) stage. Pathological staging was reclassified on the basis of the most recent (seventh) edition of the International Association for the Study of Lung Cancer Staging Project.23 The study protocol was approved by the Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea (Approval No. KC14SISI0059). Postoperative adjuvant chemotherapy with platinumbased regimens was given to patients with stage II or IIIA as a standard, and follow-up examination consisted of routine medical check-ups, including chest computed tomography at 6-month intervals for the initial 5 years and then annually thereafter. Tumor recurrence was diagnosed using radiological examinations. If possible, recurrence was confirmed cytologically and/or pathologically. The day of first detection of any recurrence was defined as the date of diagnosis of recurrence.
Formalin-fixed, paraffin wax-embedded tissue blocks of lung cancer tissue were obtained from surgical specimens. The expression of Bis protein in lung cancer tissues was evaluated by immunohistochemical staining performed on 4-lm, paraffin-wax-embedded tissue sections. Briefly, paraffin wax sections were mounted on coated slides and deparaffinized, and then rehydrated through an ethanol series. The slides were subjected to antigen retrieval with the PTLink microwave system (Dako, Glostrup, Denmark). Endogenous peroxidase was blocked with 0.3 % hydrogen peroxide for 5 min. The sections were incubated with an anti-Bis antibody (dilution 1:5000) for 30 min at room temperature, as described.8 After washing, a goat antirabbit-HRP secondary antibody (Biocare, Concord, CA) was applied and incubated for a further 20 min. The slides were visualized with Betazoid DAB Chromogen Kit (Biocare), followed by hematoxylin counterstaining.
C. D. Yeo et al. TABLE 3 Bis expression and pathological stages in patients with adenocarcinoma and squamous cell carcinoma Variable
Stage I (n = 42)
Stage II (n = 34)
Stage IIIA (n = 31)
P value
Expression intensity score Negative
30 (10–60)
0 (0–20)
10 (0–30)
0.001*
Positive (weak ? moderate ? strong)
70 (40–90)
100 (80–100)
90 (70–100)
0.001* 0.6218
Marginal activity Center, margin: equal
32 (76.2 %)
28 (82.4 %)
23 (74.2 %)
Center: negative, margin: positive
7 (16.7 %)
2 (5.9 %)
4 (12.9 %)
Center: weak, margin: strong
3 (7.1 %)
4 (11.8 %)
4 (12.9 %)
Values are numbers (percentages) for categorical variables, and medians (IQR) for continuous variables * Significant differences between stage I versus stage II ? stage IIIA, by multiple comparisons from Kruskal–Wallis test, followed by a post hoc Dunn’s test
Bis expression was assessed by two independent pathologists (GSP and GYL) using a four-tiered intensity score system (negative, weak, moderate, strong) according to the expression in adjacent nontumor tissues (Suppl. Fig. 1). Supplemental Fig. 2 shows the Bis expression in resected lung cancer tissues (adenocarcinoma and squamous cell carcinoma). Additionally, scores were expressed as percentages. Enhanced Bis expression at the periphery of a tumor facing the adjacent nontumor region was referred to as ‘‘marginal activity,’’ which was compared with the expression at the tumor center (Fig. 1). Specimens that were scored differently by the two pathologists were reexamined together to reach agreement. Statistical Analysis Demographic data are presented as numbers (%), mean ± SDs, or medians (IQR). A comparison of categorical variables was made using the v2 test or Fisher’s exact test. For continuous variables, the Wilcoxon rank-sum test was used for two groups and the Kruskal–Wallis test (with Dunn’s multiple comparison test) for more than two groups. The prognostic significance of factors affecting overall survival was determined using the Cox proportional hazard ratio (HR) model, whereas factors affecting the cumulative incidence of recurrence were determined using the semiparametric proportional hazards model for subdistribution of competing risk analysis (SAS macro ‘‘%PSHREG’’). A twosided P value \0.05 was considered to indicate statistical significance. All statistical analyses were performed using the SAS software (ver. 9.3; SAS Institute, Inc., Cary, NC). RESULTS Among the total 121 lung cancer patients, 115 were NSCLC (57 adenocarcinomas, 50 squamous cell carcinomas, 3 adenosquamous cell carcinoma, 1 poorly differentiated carcinoma with neuroendocrine differentiation, 1 large cell
neuroendocrine carcinoma, 1 mixed squamous cell carcinoma and small cell carcinoma, 1 large cell carcinoma, and 1 double primary tumor with adenocarcinoma plus squamous cell carcinoma) patients from February 2009 to December 2013, and 6 were SCLC patients from January 2002 to January 2014. Statistical analyses were performed on 107 NSCLC patients with adenocarcinomas and squamous cell carcinomas (Table 1). The mean age was 64.6 years; there were 74 (69.2 %) males and 33 (30.8 %) females. Pathological stages I, II, and IIIA were 39.3, 31.8, and 29.0 %, respectively. Table 2 shows the differences in Bis expression between adenocarcinoma and squamous cell carcinoma. Patients with squamous cell carcinomas had higher Bis expression than those with adenocarcinomas. However, marginal activity was higher in patients with adenocarcinomas than in those with squamous cell carcinomas. All six SCLC cases, one poorly differentiated carcinoma with neuroendocrine differentiation, one large cell neuroendocrine carcinoma, and one small cell carcinoma component of mixed squamous cell carcinoma and small cell carcinoma were negative for Bis expression. However, one large cell carcinoma showed strong expression of Bis protein in lung tissues (Fig. 2). Compared with stage I lung cancer, patients with stage II and IIIA lung cancer expressed higher Bis protein levels in their lung tissues (P = 0.001; Table 3). Compared with stage I adenocarcinoma, patients with stage II and IIIA adenocarcinoma expressed higher Bis protein levels (P = 0.0135; Suppl. Table 1). Among the 107 resected lung cancer patients, 38 (35.5 %) patients showed recurrence and 28 (26.2 %) died. The median relapse-free duration was 13.3 months, and median overall survival was 32.2 months. Relapse-free duration and overall survival did not differ significantly between adenocarcinomas and squamous cell carcinomas (data not shown). Moreover, recurrence and survival rate did not differ significantly according to Bis expression intensity scores or marginal activity (Suppl. Tables 2, 3).
Bis Expression in Patients with Surgically Resected Lung Cancer and its Clinical Significance
DISCUSSION The purpose of the present study was to investigate Bis expression patterns in patients with resected lung cancer and to assess its prognostic value. We observed that patients with squamous cell carcinomas had higher Bis expression than those with adenocarcinomas. However, patients with adenocarcinomas had higher marginal activity than those with squamous cell carcinomas. Bis protein was not expressed in SCLC, small cell carcinoma components of NSCLC, or NSCLC with neuroendocrine features. Higher Bis expression was also noted in patients with stage II and IIIA NSCLC than those with stage I. However, there was no significant difference in recurrence or overall survival according to Bis expression intensity scores or marginal activity. Bis is expressed ubiquitously in various tissues and is involved in the regulation of biological processes, such as apoptosis, development, cytoskeleton organization, and autophagy.24 Bis is a stress- and survival-related protein that has been shown to be upregulated in cellular stress, such as exposure to high temperature, heavy metals, proteasome inhibitors, and oxidative stress.11,14,25,26 In addition to stressed cells, in vitro, Bis is upregulated in various types of cancer and underlies resistance to therapy, through downmodulation of apoptosis.12 However, few studies related to lung cancer have been reported. Zhang et al. demonstrated that Bis and Hsp70 protein were expressed in the cytoplasm in 27.3 % (6/22) of cases of NSCLC, whereas weak Hsp70 and no Bis expression were detected in human normal bronchial epithelial cells.21 Moreover, the silencing of Bis resulted in diminished levels of Bcl-2 protein. However, the study did not evaluate the clinical characteristics of the lung cancer patients with Bis expression in lung cancer tissues or its prognostic role in recurrence or survival. Bis expression patterns in different lung cancer tissues were demonstrated using immunohistochemistry by Chiappetta et al.22 They showed Bis expression in squamous cell carcinoma was higher than that in adenocarcinoma, consistent with our results. However, they reported that 38.9 % (14/36) of SCLC cases showed no Bis expression and no Bis expression was detected in normal lung. In the present study, Bis protein was not expressed in any of the six SCLCs tested, one small cell carcinoma component of NSCLC, or two NSCLC with neuroendocrine features. However, Bis expression was detected in normal lung tissue, including type II pneumocytes, bronchial epithelial reserve cells, endothelial cells, and smooth muscle cells. One possible explanation for the discrepancy between the studies is the relatively small number of SCLC cases in our study. Additionally, we analyzed surgically resected lung tissues with pathological staging, whereas the previous study did not describe the tissue source: whether it was small tissue samples from bronchoscopic or needle
biopsies, or surgically resected tissue samples. The major concern of our study was to investigate Bis expression with clinical findings, whereas the previous study focused on the anti-apoptotic effect of Bis in SCLC. To our knowledge, this is the first report of Bis expression in lung cancer tissues according to histological classification and clinical significance. In the current study, although Bis expression was higher in squamous cell types, recurrence and survival did not differ between squamous cell carcinoma and adenocarcinoma. It has been reported that the squamous cell type of NSCLC is closely related to smoking status.27 We hypothesized that environmental stresses, such as smoking and oxidants, were more likely to promote expression of Bis protein, which is supported by high Hsp70 expression in smokers and patients with chronic obstructive pulmonary disease.28 In epithelial cell lines, a high level of Bis expression was associated with cell adhesion and migration, supporting a role for Bis in tumor invasion and metastasis.29,30 Furthermore, downmodulation of Bis decreased invasive ability in glioma cells.31 These findings are compatible with our data, showing that Bis expression was higher in patients with stage II and IIIA than those with stage I disease. Additionally, the marginal activity of Bis was significantly higher than in the center, which was more prominent in adenocarcinomas, suggesting a positive role of Bis in the motility of lung cancer cells. Regarding the molecular mechanism through which Bis modulates invasion and motility, it has been noted that Bis can interact with various signaling proteins, including CCT, guanine nucleotide exchange factor 2, and MMP-2, through the PXXP or WW domains of the Bis protein.30,32–34 However, the critical involvement of Bis in the metastasis of lung cancer should be studied using tissues from metastatic regions as well as primary sites in future studies. Our study had several limitations. First, the number of lung cancer patients was insufficient to make conclusions of clinical significance. Second, we did not measure the expression of Bcl-2, Hsp70, or Bax, important apoptotic proteins involved in the pathogenesis of Bis expression. Third, we did not consider the effect of adjuvant chemotherapy in this analysis. Cisplatin-based adjuvant chemotherapy improves survival among patients with completely resected stage II and IIIA NSCLC.35 Additionally, Bis upregulation could influence chemotherapyinduced apoptosis in neoplastic cells.26
CONCLUSIONS Our study demonstrated that Bis expression in patients with resected lung cancer differed according to histological
C. D. Yeo et al.
type and pathological stage. Further studies are needed to assess its value as a biomarker for prognosis in various types of lung cancer. ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) Grants, funded by the Korean Government (MSIP) (Nos. 2014R1A2A1A11052422, 2012R1A1A2 007589 and 2012R1A5A2047939). DISCLOSURES interests.
The authors declare that they have no competing
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