Med Oncol (2014) 31:167 DOI 10.1007/s12032-014-0167-5
ORIGINAL PAPER
Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: a comparative study Jaya Nigam • Abhijit Chandra • Hasan Raza Kazmi • Anshuman Singh • Vishal Gupta • Devendra Parmar • Manoj Kumar Srivastava
Received: 19 July 2014 / Accepted: 8 August 2014 / Published online: 17 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The role of survivin in gallbladder cancer (GBC) has not been evaluated. We investigated survivin protein expression in serum of patients with gallbladder diseases (cholelithiasis, n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Clinicopathological parameters, diagnosis and prognosis of patients with GBC were correlated with the expression of serum survivin by enzyme-linked immunosorbent assay. Significantly higher (P \ 0.0001) expression of survivin protein was observed in GBC as compared to cholelithiasis and control. Increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P \ 0.0001) and cellular differentiation (poor and moderate vs. well differentiated; P \ 0.0001) in GBC. No significant correlation was observed with any of the other clinico-pathological parameters studied. The cutoff value of survivin protein of 79 pg/ml with sensitivity of 81.16 % and specificity of 80 % differentiated the diseased group (cholelithiasis or GBC) from control group were as the cutoff value of 109 pg/ml differentiated GBC from cholelithiasis with a sensitivity of 82.05 % and specificity of 93.33 %. Though not significant, increased expression of survivin was associated with median overall survival (12 vs. 18 months; P = 0.05) in GBC patients. Our study suggests
J. Nigam A. Chandra (&) H. R. Kazmi V. Gupta Department of Surgical Gastroenterology, King George’s Medical University, Lucknow 226003, Uttar Pradesh, India e-mail: [email protected]
that survivin protein in serum could be both a useful diagnostic marker and an important prognostic factor for GBC. Keywords Cholelithiasis ELISA Gallbladder cancer Prognosis Survivin
Introduction Apoptosis is an evolutionally conserved genetic program of cellular suicide and plays an important role in organ homeostasis [1]. There is evidence that impairment of apoptosis contributes to genesis of various human malignancies. The inhibitor of apoptosis proteins (IAPs), which are widely expressed in all kinds of malignancies, plays an important role in the regulation of apoptosis. Among molecules of the IAP family, survivin, a novel anti-apoptosis gene is prominently expressed in several malignancies [2–7]. Survivin expression is correlated with more aggressive forms of disease [8, 9], poor survival [8, 10–12] and increased recurrence rates [13]. Overexpression of survivin is present not only in proliferating cancerous cells, but also in inflammatory diseases such as acute appendicitis and ulcerative colitis [14]. The imbalance between cell proliferation and cell apoptosis may be an important event in the evolution of gallbladder cancer (GBC). However, the expression and function of the survivin protein in GBC are yet not known. Therefore, we investigated the serum survivin protein expression and determined its diagnostic and prognostic relevance in GBC.
A. Singh D. Parmar Developmental Toxicology Division, Indian Institute of Toxicology Research (IITR), Lucknow, Uttar Pradesh, India
Materials and methods
M. K. Srivastava Department of Surgical Oncology, King George’s Medical University, Lucknow, Uttar Pradesh, India
The study protocol was approved by the Institutional Ethics Committee of King George’s Medical University, India,
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and was in accordance with the Helsinki Declaration. Patients with suspected GBC or cholelithiasis who were operated in the Department of Surgical Gastroenterology and General Surgery of our tertiary care center from July 2010 to Dec 2012 were enrolled in the study. Patient who were operated earlier or received prior chemo-radiotherapy or had other cancers were excluded from the study. Total 94 subjects (GBC (n = 39), cholelithiasis (n = 30) and control (n = 25)), who had given written informed consent, were studied. For GBC, only those patients were included, in whom surgical resection was performed. Diagnosis of GBC and cholelithiasis was confirmed by histopathology, and the pathological staging was done according to American Joint Committee on Cancer (AJCC) tumor node metastasis (TNM) staging 7th Edition 2010 [15]. Cholelithiasis group included cases of chronic calculus cholecystitis without any gall bladder polyp or adenoma. Control group included healthy individuals with no active inflammation, gallstones or malignancy. Peripheral blood (5 ml) was taken from subjects of each group preoperatively without any anticoagulant and was allowed to clot for 2 h at room temperature, followed by centrifugation for 20 min at 1,0009g. Serum was separated and stored in small aliquots at -20 °C until further protein analysis. ELISA Survivin protein levels were measured in serum with commercially available ELISA Kit (Uscn Life Science Inc, USA), as per the manufacturers protocol. Statistical analysis Statistical analysis was done using GraphPad Prism version 5.0 (GraphPad Software, Inc., California, USA). Multiple groups were analyzed using one-way analysis of variance (ANOVA) test. Unpaired Student’s t test was used for comparing two groups. The Receiver operating characteristic curve (ROC) was constructed to see the diagnostic utility and cutoff value of the test. Survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. The significance of various parameters for survival was analyzed by the Cox proportional hazards model. Two-tailed p value \0.05 was considered statistically significant.
Results The general characteristics of subjects participated in the study are given in Table 1. The mean – SE value of survivin protein was 139.02 – 4.02 in GBC patients. This was
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significantly higher (P \ 0.0001) than in cholelithiasis (82.98 – 3.38) group and healthy control (70.64 – 2.38) group. However, difference of survivin level between patients with cholelithiasis and healthy volunteers was not statistically significant (Table 2). In GBC patients, significantly higher expression of survivin was observed in stage III as compared to stage II (P \ 0.0001) and poorly differentiated tumors as compared to well differentiated ones (P \ 0.0001) (Table 3). However no significant association was found between survivin protein expression and other clinicopathological variables like age, gender and presence or absence of gallstones for GBC patients (Table 3). A receiver operator characteristics (ROC) curve was constructed, by plotting sensitivity versus 100-specificity, and was used to determine an optimal cutoff values, which best segregates diseased (cholelithiasis or GBC) from nondiseased and GBC from cholelithiasis individuals. The cutoff value of this marker for diseased group was found to be 79 pg/ml. The area under the ROC curve was 0.865, corresponding to a sensitivity of 81.16 % (95 % CI 69.94–89.51) and specificity of 80 % (95 % CI 59.30–93.17) (Fig. 1a). Whereas the cutoff value for GBC was 109 pg/ml, yielding area under curve 0.944, sensitivity of 82.05 % (95 % CI 66.46–92.46) and specificity of 93.33 % (95 % CI 77.93–99.18) in differentiating GBC from cholelithiasis (Fig. 1b). To elucidate the prognostic role of survivin protein expression in GBC patients, we examined the relationship Table 1 Characteristics of control, cholelithiasis and gallbladder cancer (GBC) patients Parameters
Control (n = 25)
Cholelithiasis (n = 30)
GBC (n = 39)
Age (Mean – SD) (years)
41.80 – 13.16
40.73 – 12.34
47.30 – 12.20
(Range)
(19–63)
(15–65)
(21–67)
Male
11
12
12
Female
14
18
27
Gender
Gallstone Present
0
All
28
Absent
All
0
11
–
–
22
–
–
17
Stage II III Cellular differentiation Well
–
–
19
Poor and moderate
–
–
20
SD standard deviation
Med Oncol (2014) 31:167
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Table 2 Survivin protein concentration in serum of control, cholelithiasis and GBC patients as determined by ELISA Group (no. of cases)
Survivin in pg/ml Range
ANOVA Mean – SE
Tukey’s Multiple comparison
P value
Control (n = 25)
50–90
70.64 – 2.38
Cholelithiasis (n = 30)
59–121
82.98 – 3.38
GBC (n = 39)
83–165
139.02 – 4.02
\0.0001
Control vs. Cholelithasis (NS) Control vs. GBC* Cholelithiasis vs. GBC*
Data are represented as mean – standard error (SE). The statistical difference between the samples was analyzed by nonparametric test (ANOVA and Tukey’s post hoc test) NS nonsignificant, GBC gallbladder cancer * Indicates statistically significant difference (P \ 0.05)
Table 3 Correlation of survivin protein with various clinicopathological factors for gallbladder cancer (GBC) patients Parameters
Mean (pg/ml) ± SE
P value
Age (years) B47 (n = 16)
134.2 – 5.49
[47 (n = 23)
146.0 – 5.53
0.1501
Gender Male (n = 12)
141.4 – 8.15
Female (n = 27)
138.0 – 4.64
0.7042
Gallstone Present (n = 28)
135.1 – 5.07
Absent (n = 11)
149.0 – 5.21
0.1220
Stage II (n = 22)
123.1 – 4.86
III (n = 17)
159.7 – 0.82
\0.0001*
Cellular differentiation Well (n = 19) Poor and moderate (n = 20)
119.4 – 5.14 157.7 – 1.32
Fig. 2 Kaplan–Meier overall survival curve of gallbladder cancer patients (n = 39) in relation to survivin protein expression. The survival of patients with higher survivin expression was significantly poor (P = 0.057)
\0.0001*
* Indicates statistically significant difference (P \ 0.05)
between survivin expression and patient survival. The median value of survivin protein was 149.82 pg/ml for GBC patients. Therefore, patients were divided into two groups: one with protein value [149.82 pg/ml and other with protein value B149.82 pg/ml. The Kaplan–Meier
survival curve demonstrated that patients with higher survivin expression had shorter survival than those with low survivin protein expression (median overall survival 12 vs. 18 months) (HR 0.4802, 95 % CI 0.225–1.025; P = 0.057) (Fig. 2).
Discussion Gallbladder cancer is a highly fatal malignancy of the biliary tract. The etiology of this tumor is complex, though
Fig. 1 ROC curves of serum survivin in terms of sensitivity and specificity which predicts: a the presence of gallbladder disease AUC = 0.865 (86.5 %), P \ 0.0001. b the presence of gallbladder cancers AUC = 0.944 (94.4 %), P \ 0.0001
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there is a strong association with cholelithiasis (gallstones). Its poor prognosis is attributed to late diagnosis due to absence of specific clinical findings in early stages and lack of biomarkers for early identification [16–18]. Newer diagnostic and prognostic markers are needed for early diagnosis and management of this lethal disease. To the best of our knowledge, this is the first study to comprehensively analyze the survivin protein expression in the serum of GBC patients and provide an initial investigation of its potential as a diagnostic and prognostic biomarker for this dreaded disease. Survivin was earlier viewed as an intracellular molecule that exists in subcellular locations, such as cytoplasm, nucleus and mitochondria. Currently, survivin (mRNA or protein) has also been shown in extracellular space, such as cell cultures and certain body fluids [19, 20]. In the present study, we found significantly elevated serum levels of survivin protein in GBC as compared to cholelithiasis and control. Our study showed that survivin might play an important role in tumorigenesis and progression of GBC, as survivin protein levels in the serum of GBC patients were significantly related with tumor differentiation, and tumor stage. Similar findings were noticed by Kappler et al. [21] and Lee et al. [22] in soft tissue sarcoma and gastric cancer, respectively. Detection of survivin in body fluids may serve as a diagnostic marker allowing early detection of malignancy and might effectively discriminate malignant from benign conditions [23–25]. In line with these findings, our results also demonstrated that the detection of survivin protein in serum has very good sensitivity and specificity in screening individuals with gallbladder disease (cholelithiasis or GBC) and also in discriminating GBC from cholelithiasis. This indicates that serum survivin can be a useful molecular marker for the diagnosis of GBC. Cholelithiasis is an important risk factor for the development of GBC [17]. However, no association was observed either for the presence or absence of cholelithiasis in GBC. This may be due to the small number of GBC patients studied for the same. Numerous studies have appeared over the past several years indicating the prognostic importance of survivin expression at both the mRNA and protein level [26–28]. To identify the potential prognostic value of serum survivin protein for GBC, the associations between survivin protein level and survival were evaluated in this study. Patients with high levels of survivin protein had a poor overall survival compared with those with low levels (12 vs. 18 months); however, this difference was not significant (P = 0.05). These discordant results may be due to small sample size and shorter survival of GBC patients. Therefore, further studies with larger sample size are required in this direction.
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The current study has a limitation of stage I and IV GBC samples. Our center being a tertiary referral institute, we do not experience patients of stage I GBC as most of such cases are detected incidentally during cholecystectomy performed for benign diseases at others centers, limiting preoperative sample acquisition. Patients who were not considered for definitive surgery (stage IV) were excluded as per the study protocol. In conclusion, our data suggest that over-expression of survivin protein in serum could be a useful marker for noninvasive tumor detection and an important prognostic factor for GBC. Acknowledgments This work was supported by the Indian Council of Medical Research, New Delhi, India (Grant No. 3/2/2/130/2012/ NCD-III). Conflict of interest
None.
References 1. Vaux DL, Korsmeyer SJ. Cell death in development. Cell. 1999;96(2):245–54. 2. Takai N, Miyazaki T, Nishida M, Nasu K, Miyakawa I. Survivin expression correlates with clinical stage, histological grade, invasive behavior and survival rate in endometrial carcinoma. Cancer Lett. 2002;184(1):105–16. 3. Monzo´ M, Rosell R, Felip E, Astudillo J, Sa´nchez JJ, Maestre J, et al. A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol. 1999;17(7):2100–4. 4. Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000;6(1):127–34. 5. Ikeguchi M, Kaibara N. Survivin messenger RNA expression is a good prognostic biomarker for oesophageal carcinoma. Br J Cancer. 2002;87(8):883–7. 6. Ikeguchi M, Hirooka Y, Kaibara N. Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma. Cancer. 2002;95(9):1938–45. 7. Asanuma K, Moriai R, Yajima T, Yagihashi A, Yamada M, Kobayashi D, et al. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res. 2000;91(11):1204–9. 8. Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet. 1998;351(9106):882–3. 9. Lo Muzio L, Staibano S, Pannone G, Mignogna MD, Mariggio` A, Salvatore G, et al. Expression of the apoptosis inhibitor survivin in aggressive squamous cell carcinoma. Exp Mol Pathol. 2001;70(3):249–54. 10. Monzo´ M, Rosell R, Felip E, Astudillo J, Sa´nchez JJ, Maestre J, et al. A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol. 1999;17(7):2100–4. 11. Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998;58(22):5071–4. 12. Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000;6(1):127–34.
Med Oncol (2014) 31:167 13. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclindependent kinases. Genes Dev. 1995;9(10):1149–63. 14. Altznauer F, Martinelli S, Yousefi S, Thu¨rig C, Schmid I, Conway EM, et al. Inflammation-associated cell cycle-independent block of apoptosis by survivin in terminally differentiated neutrophils. J Exp Med. 2004;199(10):1343–54. 15. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 2010;17(6):1471–4. 16. Shukla HS, Awasthi K, Naithani YP, Gupta SC. A clinico-pathological study of carcinoma of the gall bladder. Indian J Cancer. 1981;18(3):198–201. 17. Misra S, Chaturvedi A, Misra NC, Sharma ID. Carcinoma of the gallbladder. Lancet Oncol. 2003;4(3):167–76. 18. Kazmi HR, Chandra A, Nigam J, Noushif M, Parmar D, Gupta V. Prognostic significance of K-ras codon 12 mutation in patients with resected gallbladder cancer. Dig Surg. 2013;30(3):233–9. 19. Schwarzenbach H, Hoon DS, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. 2011;11(6):426–37. 20. Khan S, Jutzy JM, Valenzuela MM, Turay D, Aspe JR, Ashok A, et al. Plasma-derived exosomal survivin, a plausible biomarker for early detection of prostate cancer. PLoS One. 2012;7(10): e46737. 21. Kappler M, Kotzsch M, Bartel F, Fu¨ssel S, Lautenschla¨ger C, Schmidt U, et al. Elevated expression level of survivin protein in soft-tissue sarcomas is a strong independent predictor of survival. Clin Cancer Res. 2003;9(3):1098–104.
Page 5 of 5 167 22. Lee GH, Joo YE, Koh YS, Chung IJ, Park YK, Lee JH, et al. Expression of survivin in gastric cancer and its relationship with tumor angiogenesis. Eur J Gastroenterol Hepatol. 2006;18(9): 957–63. 23. Wang T, Qian X, Wang Z, Wang L, Yu L, Ding Y, et al. Detection of cell-free BIRC5 mRNA in effusions and its potential diagnostic value for differentiating malignant and benign effusions. Int J Cancer. 2009;125(8):1921–5. 24. Smith SD, Wheeler MA, Plescia J, Colberg JW, Weiss RM, Altieri DC. Urine detection of survivin and diagnosis of bladder cancer. JAMA. 2001;285(3):324–8. 25. Wu YK, Chen KT, Kuo YB, Huang YS, Chan EC. Quantitative detection of survivin in malignant pleural effusion for the diagnosis and prognosis of lung cancer. Cancer Lett. 2009;273(2): 331–5. 26. Wang TT, Qian XP, Liu BR. Survivin: potential role in diagnosis, prognosis and targeted therapy of gastric cancer. World J Gastroenterol. 2007;13(20):2784–90. 27. Nigam J, Chandra A, Kazmi HR, Parmar D, Singh D, Gupta V, et al. Expression of survivin mRNA in gallbladder cancer: a diagnostic and prognostic marker? Tumour Biol. 2014. doi:10. 1007/s13277-014-2200-x. 28. Chen P, Zhu J, Liu DY, Li HY, Xu N, Hou M. Over-expression of survivin and VEGF in small-cell lung cancer may predict the poorer prognosis. Med Oncol. 2014;31(1):775.
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ORIGINAL PAPER
Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: a comparative study Jaya Nigam • Abhijit Chandra • Hasan Raza Kazmi • Anshuman Singh • Vishal Gupta • Devendra Parmar • Manoj Kumar Srivastava
Received: 19 July 2014 / Accepted: 8 August 2014 / Published online: 17 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The role of survivin in gallbladder cancer (GBC) has not been evaluated. We investigated survivin protein expression in serum of patients with gallbladder diseases (cholelithiasis, n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Clinicopathological parameters, diagnosis and prognosis of patients with GBC were correlated with the expression of serum survivin by enzyme-linked immunosorbent assay. Significantly higher (P \ 0.0001) expression of survivin protein was observed in GBC as compared to cholelithiasis and control. Increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P \ 0.0001) and cellular differentiation (poor and moderate vs. well differentiated; P \ 0.0001) in GBC. No significant correlation was observed with any of the other clinico-pathological parameters studied. The cutoff value of survivin protein of 79 pg/ml with sensitivity of 81.16 % and specificity of 80 % differentiated the diseased group (cholelithiasis or GBC) from control group were as the cutoff value of 109 pg/ml differentiated GBC from cholelithiasis with a sensitivity of 82.05 % and specificity of 93.33 %. Though not significant, increased expression of survivin was associated with median overall survival (12 vs. 18 months; P = 0.05) in GBC patients. Our study suggests
J. Nigam A. Chandra (&) H. R. Kazmi V. Gupta Department of Surgical Gastroenterology, King George’s Medical University, Lucknow 226003, Uttar Pradesh, India e-mail: [email protected]
that survivin protein in serum could be both a useful diagnostic marker and an important prognostic factor for GBC. Keywords Cholelithiasis ELISA Gallbladder cancer Prognosis Survivin
Introduction Apoptosis is an evolutionally conserved genetic program of cellular suicide and plays an important role in organ homeostasis [1]. There is evidence that impairment of apoptosis contributes to genesis of various human malignancies. The inhibitor of apoptosis proteins (IAPs), which are widely expressed in all kinds of malignancies, plays an important role in the regulation of apoptosis. Among molecules of the IAP family, survivin, a novel anti-apoptosis gene is prominently expressed in several malignancies [2–7]. Survivin expression is correlated with more aggressive forms of disease [8, 9], poor survival [8, 10–12] and increased recurrence rates [13]. Overexpression of survivin is present not only in proliferating cancerous cells, but also in inflammatory diseases such as acute appendicitis and ulcerative colitis [14]. The imbalance between cell proliferation and cell apoptosis may be an important event in the evolution of gallbladder cancer (GBC). However, the expression and function of the survivin protein in GBC are yet not known. Therefore, we investigated the serum survivin protein expression and determined its diagnostic and prognostic relevance in GBC.
A. Singh D. Parmar Developmental Toxicology Division, Indian Institute of Toxicology Research (IITR), Lucknow, Uttar Pradesh, India
Materials and methods
M. K. Srivastava Department of Surgical Oncology, King George’s Medical University, Lucknow, Uttar Pradesh, India
The study protocol was approved by the Institutional Ethics Committee of King George’s Medical University, India,
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and was in accordance with the Helsinki Declaration. Patients with suspected GBC or cholelithiasis who were operated in the Department of Surgical Gastroenterology and General Surgery of our tertiary care center from July 2010 to Dec 2012 were enrolled in the study. Patient who were operated earlier or received prior chemo-radiotherapy or had other cancers were excluded from the study. Total 94 subjects (GBC (n = 39), cholelithiasis (n = 30) and control (n = 25)), who had given written informed consent, were studied. For GBC, only those patients were included, in whom surgical resection was performed. Diagnosis of GBC and cholelithiasis was confirmed by histopathology, and the pathological staging was done according to American Joint Committee on Cancer (AJCC) tumor node metastasis (TNM) staging 7th Edition 2010 [15]. Cholelithiasis group included cases of chronic calculus cholecystitis without any gall bladder polyp or adenoma. Control group included healthy individuals with no active inflammation, gallstones or malignancy. Peripheral blood (5 ml) was taken from subjects of each group preoperatively without any anticoagulant and was allowed to clot for 2 h at room temperature, followed by centrifugation for 20 min at 1,0009g. Serum was separated and stored in small aliquots at -20 °C until further protein analysis. ELISA Survivin protein levels were measured in serum with commercially available ELISA Kit (Uscn Life Science Inc, USA), as per the manufacturers protocol. Statistical analysis Statistical analysis was done using GraphPad Prism version 5.0 (GraphPad Software, Inc., California, USA). Multiple groups were analyzed using one-way analysis of variance (ANOVA) test. Unpaired Student’s t test was used for comparing two groups. The Receiver operating characteristic curve (ROC) was constructed to see the diagnostic utility and cutoff value of the test. Survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. The significance of various parameters for survival was analyzed by the Cox proportional hazards model. Two-tailed p value \0.05 was considered statistically significant.
Results The general characteristics of subjects participated in the study are given in Table 1. The mean – SE value of survivin protein was 139.02 – 4.02 in GBC patients. This was
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significantly higher (P \ 0.0001) than in cholelithiasis (82.98 – 3.38) group and healthy control (70.64 – 2.38) group. However, difference of survivin level between patients with cholelithiasis and healthy volunteers was not statistically significant (Table 2). In GBC patients, significantly higher expression of survivin was observed in stage III as compared to stage II (P \ 0.0001) and poorly differentiated tumors as compared to well differentiated ones (P \ 0.0001) (Table 3). However no significant association was found between survivin protein expression and other clinicopathological variables like age, gender and presence or absence of gallstones for GBC patients (Table 3). A receiver operator characteristics (ROC) curve was constructed, by plotting sensitivity versus 100-specificity, and was used to determine an optimal cutoff values, which best segregates diseased (cholelithiasis or GBC) from nondiseased and GBC from cholelithiasis individuals. The cutoff value of this marker for diseased group was found to be 79 pg/ml. The area under the ROC curve was 0.865, corresponding to a sensitivity of 81.16 % (95 % CI 69.94–89.51) and specificity of 80 % (95 % CI 59.30–93.17) (Fig. 1a). Whereas the cutoff value for GBC was 109 pg/ml, yielding area under curve 0.944, sensitivity of 82.05 % (95 % CI 66.46–92.46) and specificity of 93.33 % (95 % CI 77.93–99.18) in differentiating GBC from cholelithiasis (Fig. 1b). To elucidate the prognostic role of survivin protein expression in GBC patients, we examined the relationship Table 1 Characteristics of control, cholelithiasis and gallbladder cancer (GBC) patients Parameters
Control (n = 25)
Cholelithiasis (n = 30)
GBC (n = 39)
Age (Mean – SD) (years)
41.80 – 13.16
40.73 – 12.34
47.30 – 12.20
(Range)
(19–63)
(15–65)
(21–67)
Male
11
12
12
Female
14
18
27
Gender
Gallstone Present
0
All
28
Absent
All
0
11
–
–
22
–
–
17
Stage II III Cellular differentiation Well
–
–
19
Poor and moderate
–
–
20
SD standard deviation
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Table 2 Survivin protein concentration in serum of control, cholelithiasis and GBC patients as determined by ELISA Group (no. of cases)
Survivin in pg/ml Range
ANOVA Mean – SE
Tukey’s Multiple comparison
P value
Control (n = 25)
50–90
70.64 – 2.38
Cholelithiasis (n = 30)
59–121
82.98 – 3.38
GBC (n = 39)
83–165
139.02 – 4.02
\0.0001
Control vs. Cholelithasis (NS) Control vs. GBC* Cholelithiasis vs. GBC*
Data are represented as mean – standard error (SE). The statistical difference between the samples was analyzed by nonparametric test (ANOVA and Tukey’s post hoc test) NS nonsignificant, GBC gallbladder cancer * Indicates statistically significant difference (P \ 0.05)
Table 3 Correlation of survivin protein with various clinicopathological factors for gallbladder cancer (GBC) patients Parameters
Mean (pg/ml) ± SE
P value
Age (years) B47 (n = 16)
134.2 – 5.49
[47 (n = 23)
146.0 – 5.53
0.1501
Gender Male (n = 12)
141.4 – 8.15
Female (n = 27)
138.0 – 4.64
0.7042
Gallstone Present (n = 28)
135.1 – 5.07
Absent (n = 11)
149.0 – 5.21
0.1220
Stage II (n = 22)
123.1 – 4.86
III (n = 17)
159.7 – 0.82
\0.0001*
Cellular differentiation Well (n = 19) Poor and moderate (n = 20)
119.4 – 5.14 157.7 – 1.32
Fig. 2 Kaplan–Meier overall survival curve of gallbladder cancer patients (n = 39) in relation to survivin protein expression. The survival of patients with higher survivin expression was significantly poor (P = 0.057)
\0.0001*
* Indicates statistically significant difference (P \ 0.05)
between survivin expression and patient survival. The median value of survivin protein was 149.82 pg/ml for GBC patients. Therefore, patients were divided into two groups: one with protein value [149.82 pg/ml and other with protein value B149.82 pg/ml. The Kaplan–Meier
survival curve demonstrated that patients with higher survivin expression had shorter survival than those with low survivin protein expression (median overall survival 12 vs. 18 months) (HR 0.4802, 95 % CI 0.225–1.025; P = 0.057) (Fig. 2).
Discussion Gallbladder cancer is a highly fatal malignancy of the biliary tract. The etiology of this tumor is complex, though
Fig. 1 ROC curves of serum survivin in terms of sensitivity and specificity which predicts: a the presence of gallbladder disease AUC = 0.865 (86.5 %), P \ 0.0001. b the presence of gallbladder cancers AUC = 0.944 (94.4 %), P \ 0.0001
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there is a strong association with cholelithiasis (gallstones). Its poor prognosis is attributed to late diagnosis due to absence of specific clinical findings in early stages and lack of biomarkers for early identification [16–18]. Newer diagnostic and prognostic markers are needed for early diagnosis and management of this lethal disease. To the best of our knowledge, this is the first study to comprehensively analyze the survivin protein expression in the serum of GBC patients and provide an initial investigation of its potential as a diagnostic and prognostic biomarker for this dreaded disease. Survivin was earlier viewed as an intracellular molecule that exists in subcellular locations, such as cytoplasm, nucleus and mitochondria. Currently, survivin (mRNA or protein) has also been shown in extracellular space, such as cell cultures and certain body fluids [19, 20]. In the present study, we found significantly elevated serum levels of survivin protein in GBC as compared to cholelithiasis and control. Our study showed that survivin might play an important role in tumorigenesis and progression of GBC, as survivin protein levels in the serum of GBC patients were significantly related with tumor differentiation, and tumor stage. Similar findings were noticed by Kappler et al. [21] and Lee et al. [22] in soft tissue sarcoma and gastric cancer, respectively. Detection of survivin in body fluids may serve as a diagnostic marker allowing early detection of malignancy and might effectively discriminate malignant from benign conditions [23–25]. In line with these findings, our results also demonstrated that the detection of survivin protein in serum has very good sensitivity and specificity in screening individuals with gallbladder disease (cholelithiasis or GBC) and also in discriminating GBC from cholelithiasis. This indicates that serum survivin can be a useful molecular marker for the diagnosis of GBC. Cholelithiasis is an important risk factor for the development of GBC [17]. However, no association was observed either for the presence or absence of cholelithiasis in GBC. This may be due to the small number of GBC patients studied for the same. Numerous studies have appeared over the past several years indicating the prognostic importance of survivin expression at both the mRNA and protein level [26–28]. To identify the potential prognostic value of serum survivin protein for GBC, the associations between survivin protein level and survival were evaluated in this study. Patients with high levels of survivin protein had a poor overall survival compared with those with low levels (12 vs. 18 months); however, this difference was not significant (P = 0.05). These discordant results may be due to small sample size and shorter survival of GBC patients. Therefore, further studies with larger sample size are required in this direction.
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The current study has a limitation of stage I and IV GBC samples. Our center being a tertiary referral institute, we do not experience patients of stage I GBC as most of such cases are detected incidentally during cholecystectomy performed for benign diseases at others centers, limiting preoperative sample acquisition. Patients who were not considered for definitive surgery (stage IV) were excluded as per the study protocol. In conclusion, our data suggest that over-expression of survivin protein in serum could be a useful marker for noninvasive tumor detection and an important prognostic factor for GBC. Acknowledgments This work was supported by the Indian Council of Medical Research, New Delhi, India (Grant No. 3/2/2/130/2012/ NCD-III). Conflict of interest
None.
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