European Journal of Cardiovascular Medicine

Gallbladder cancer (GBC) is a rare but highly aggressive malignancy with a poor prognosis, often diagnosed at advanced stages due to its asymptomatic early course and nonspecific clinical presentation [1, 2]. It is the most common biliary tract cancer and ranks among the leading causes of cancer-related deaths in certain high-risk regions, including parts of South America, Eastern Europe, and Southeast Asia [3]. The five-year survival rate remains dismal, ranging from 5% to 15%, primarily due to late diagnosis and limited therapeutic [4]. Early detection and accurate prognostic assessment are crucial for improving clinical outcomes, yet reliable biomarkers for GBC remain an unmet need.

Current diagnostic approaches for GBC rely heavily on imaging techniques such as ultrasonography, computed tomography (CT), and magnetic resonance imaging (MRI), which often detect the disease at advanced stages [5]. Tissue biopsy remains the gold standard for definitive diagnosis, but its invasiveness and potential complications limit its utility in early screening [6]. Serum tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), have been explored for their diagnostic and prognostic value in GBC, but their sensitivity and specificity are suboptimal [7]. Thus, there is an urgent need to identify novel biomarkers that can enhance early detection and predict disease progression more accurately.

CEA and CA 19-9 are the most extensively studied tumor markers in GBC, with elevated levels frequently observed in advanced disease [7]. However, their utility is limited by false-positive elevations in benign biliary conditions such as cholangitis and obstructive jaundice. Additionally, CA 19-9 is not expressed in individuals with Lewis antigen-negative blood groups, further restricting its diagnostic applicability [8-12]. Given these limitations, the search for complementary biomarkers with higher specificity and prognostic relevance is essential to improve clinical decision-making in GBC management.

Survivin, a member of the inhibitor of apoptosis (IAP) protein family, has emerged as a promising biomarker in various cancers due to its role in regulating cell proliferation and apoptosis [13]. Overexpression of survivin has been linked to tumor aggressiveness, chemotherapy resistance, and poor survival outcomes in multiple malignancies, including gastrointestinal cancers [13]. In GBC, survivin expression has been associated with advanced tumor stage, lymph node metastasis, and reduced overall survival [13]. However, its diagnostic and prognostic significance in comparison to conventional markers like CEA and CA 19-9 remains underexplored.

The present study aims to evaluate the diagnostic and prognostic performance of CEA, CA 19-9, and survivin in GBC patients, comparing their sensitivity, specificity, and correlation with clinicopathological features. By assessing these biomarkers in serum and tissue samples, we seek to determine their individual and combined utility in distinguishing GBC from benign gallbladder diseases and predicting disease outcomes.

This was an “observational & prospective study” conducted in a “tertiary care hospital of eastern India” from January 2024 to December 2024. The study was done on patients of gall bladder carcinoma who gave their consent to participate in the study after being aware of their rights and obligations in research as per “Declaration of Helsinki & Good Clinical Practice”.

Sample Size: Consecutive sampling was done and all patients of gall bladder carcinoma meeting our inclusion and exclusion criteria were included in our study. Equal number of age and gender matched healthy controls were included.

Inclusion Criteria:

• Patients with pathologically confirmed gallbladder carcinoma (GBC).
• Patients who underwent surgical resection with curative intent.
• Availability of complete clinical and pathological data for analysis.

Exclusion Criteria:

• Patients with metastatic gallbladder carcinoma.
• Patients who received neoadjuvant therapy before surgery.
• Cases with unclear disease diagnosis.
• Patients with incomplete clinical data.
• Patients with a history of other malignancies.
• Those who experienced perioperative death.
• Patients lost to follow-up shortly after discharge.

Serum level of “tumour markers CEA and CA19.9” were estimated by “chemiluminescent immunoassay (CLIA)” method in department of Biochemistry, maintaining all the quality control, calibrator and reagent kit. “Survivin protein” level in serum was estimated by “commercially available ELISA Kit.”

Clinical and pathological data were collected, including “age, gender, presence of gallstones, preoperative (within one week of surgery) alpha fetoprotein (AFP), CEA, and CA19-9, tumour location, pathology, presence of jaundice, type of resection, extrahepatic bile duct resection, the American Joint Commission on Cancer (AJCC) staging system, tumour differentiation, survival time, survival status” [14]. “Serum total bilirubin >34 μmol/L” before the operation was defined as jaundice [15]. “AFP >20 μg/L, CEA >5 ng/ml, Survivin >109 pg/ml and CA19-9 >37 U/mL” were the upper limits of normal [7, 16].

"Gallbladder en bloc resection and regional lymph node dissection" were standard procedures.  The partial hepatectomy included an extended right/left hepatectomy and "wedge resection with a 2 cm margin" (containing segments IVb/V).  With the hope of achieving a R0 resection, a simultaneous resection of "the bile duct, duodenum, stomach, colon, omentum, and/or pancreatic head" was carried out if the GBC had infiltrated the surrounding organs [15].  R0 resection in this study was characterized as "pathologically negative procedural margins," while R1 and R2 showed "microscopic and gross residual disease", respectively [17].  

50 patients with gall bladder cancer undergoing surgical resection and 50 controls (age and gender matched) were included in the study. 

Their demographic and clinical characteristics of patients with Gall Bladder Cancer are given in Table 1.

Table 1: Demographic and Clinical Characteristics of Patients with GBC

The study observed 50 gall bladder cancer (GBC) patients, with 36% being male. A small proportion (6%) had elevated AFP (>20 µg/L). Bile duct resection was performed in 24% of cases, while liver resection was remarkably common at 94%. Resection margins showed that 72% achieved R0 (complete resection), whereas 28% had R1 (microscopic residual tumor). Advanced AJCC grading (III-IV) was predominant in 92% of patients, indicating later-stage cancer, and the average age was approximately 58.64 years [Table 1].

Table 2: Comparison of CEA between Cases (GBC) and Controls

Carcinoembryonic antigen (CEA) levels over 5 ng/ml were more frequent in GBC cases (52%) compared to controls (26%). The test showed a sensitivity of 52% and specificity of 74%, indicating moderate diagnostic accuracy. The positive likelihood ratio (2.0) and negative likelihood ratio (0.65) further suggest that elevated CEA levels have limited predictive value for GBC [Table 2]. 

Table 3: Comparison of CA19-9 between Cases (GBC) and Controls

A higher percentage of GBC cases (64%) had elevated CA19-9 (>37 U/ml) compared to controls (22%). The sensitivity (64%) and specificity (78%) were better than for CEA, with a positive likelihood ratio of 2.91 and a negative likelihood ratio of 0.46, indicating CA19-9 has moderate reliability as a diagnostic marker [Table 3].              

Table 4: Comparison of Survivin between Cases (GBC) and Controls

Survivin levels (>109 pg/ml) were markedly higher in GBC cases (82%) than in controls (6%). The test demonstrated high sensitivity (82%) and specificity (94%), making it a strong candidate for GBC diagnosis. The positive likelihood ratio (13.67) and negative likelihood ratio (0.19) further reinforce its diagnostic utility [Table 4].

Table 5: Correlation of Survival in GBC Patients with CEA, CA19-9, and Survivin

Survival outcomes correlated with marker levels. Patients with elevated markers (CEA >5 ng/ml, CA19-9 >37 U/ml, and Survivin >109 pg/ml) had shorter survival durations, with ≤12-month survival being more common. Conversely, patients with lower levels of these markers generally showed longer survival (>12 months), suggesting their potential prognostic value [Table 5].

The study focused on evaluating CEA, CA19-9, and survivin as potential biomarkers for GBC. Survivin, a member of the inhibitor of apoptosis protein family, has emerged as a promising candidate due to its role in regulating cell proliferation and apoptosis. Overexpression of survivin has been linked to tumor aggressiveness and poor outcomes in various cancers, including gastrointestinal malignancies. The rationale for comparing these markers lies in their distinct biological roles: CEA is associated with cell adhesion and metastasis, CA19-9 reflects tumor burden, and survivin is implicated in cancer cell survival and resistance to apoptosis. By assessing their diagnostic and prognostic performance, the study aimed to identify a more effective tool for managing GBC.

The results revealed significant differences in the diagnostic accuracy of the three markers. CEA demonstrated moderate sensitivity (52%) and specificity (74%), consistent with its known limitations in distinguishing GBC from benign conditions. CA19-9 performed better, with a sensitivity of 64% and specificity of 78%, but its diagnostic value was still constrained by its variability in non-malignant diseases. In contrast, survivin stood out with remarkably high sensitivity (82%) and specificity (94%), making it a strong candidate for GBC diagnosis. The positive and negative likelihood ratios further reinforced survivin’s diagnostic utility, suggesting it could significantly reduce false positives and improve early detection rates.

Beyond diagnosis, the study also highlighted the prognostic significance of these markers. Elevated levels of CEA, CA19-9, and survivin were associated with shorter survival durations (≤12 months), with survivin showing the strongest correlation. Notably, none of the patients with low survivin levels (≤109 pg/ml) died within 12 months, whereas 17% of those with high levels had poor outcomes. This suggests that survivin could serve as a valuable tool for risk stratification, helping clinicians identify patients who may benefit from more aggressive treatment or closer monitoring. The combination of survivin with conventional markers like CA19-9 could further enhance prognostic accuracy, offering a more comprehensive assessment of disease progression.

Our findings align closely with Nigam et al. (2014), who reported that survivin levels were significantly elevated in GBC patients (cutoff: 109 pg/ml) with a sensitivity of 82.05% and specificity of 93.33%, mirroring our results (82% sensitivity, 94% specificity) [18]. Both studies confirm survivin’s strong diagnostic potential in distinguishing GBC from benign conditions. Additionally, Nigam et al. observed reduced median survival (12 vs. 18 months) in patients with high survivin, consistent with our prognostic data where elevated survivin correlated with ≤12-month survival [18].

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However, Gupta et al. (2016) reported survivin overexpression in GBC but found no significant correlation with tumor stage or differentiation [19], contrasting our results and those of Nigam et al. This discrepancy may stem from methodological differences (immunohistochemistry vs. serum ELISA in our study) or smaller sample sizes. Salman et al. (2016) supported survivin’s prognostic role, linking its expression to reduced survival (p = 0.043), reinforcing our conclusion that survivin is a valuable prognostic marker [20].

Our study found moderate sensitivity (52%) and specificity (74%) for CEA, consistent with Strom et al. (1990), who reported 50% sensitivity and 92.7% specificity at a 4.0 ng/mL cutoff. The higher specificity in Strom’s study may reflect differences in control groups (gallstones vs. healthy controls in our study) [21]. For CA19-9, our results (64% sensitivity, 78% specificity) were comparable to Strom et al. (79.4% sensitivity, 79.2% specificity at 20 U/mL) but lower than Sachan et al. (2020), who reported 52% sensitivity and 80% specificity at a higher cutoff (72 U/mL) [21, 22]. This suggests that CA19-9’s diagnostic accuracy may vary with cutoff selection and patient cohorts.

Prognostically, our data corroborate Wen et al. (2017) and Kim et al. (2021), who identified elevated CA19-9 and CEA as independent predictors of poor survival. Wen et al. noted a median survival of 6 months for patients with both markers elevated, similar to our observation of shorter survival in such cases. Kim et al. further validated CA19-9’s superiority over CEA (HR: 2.557 vs. 1.613), aligning with our finding that CA19-9 had better prognostic discrimination [23, 24]. Yu et al. (2014) also emphasized the combined use of CA19-9 and CEA for survival stratification, with our study reinforcing this approach [25].

Liu et al. (2011) demonstrated survivin’s therapeutic potential by targeting it with an oncolytic adenovirus (AdSurp-P53), achieving significant tumor regression in GBC models [26]. Our study’s strong association between survivin and poor outcomes supports survivin’s role as a therapeutic target, suggesting that strategies like gene therapy or survivin inhibitors could be explored clinically.

The clinical implications of these findings are substantial. Survivin’s high specificity could aid in differentiating GBC from benign gallbladder diseases, facilitating earlier intervention and improving patient outcomes. Its prognostic value may also refine current staging systems, such as the AJCC criteria, by incorporating biomarker data to better predict survival and guide therapeutic decisions. Additionally, the role of survivin in apoptosis resistance raises the possibility of targeting it therapeutically, opening new avenues for research into novel treatments for GBC.

However, the study’s small sample size and single-center design limit the generalizability of the findings. Larger, multicenter studies are needed to validate these results and explore survivin’s dynamics during treatment. Further research into the mechanistic role of survivin in GBC pathogenesis could also provide insights for developing targeted therapies.

In conclusion, this study demonstrates that survivin outperforms CEA and CA19-9 as a diagnostic and prognostic marker for GBC. Its integration into clinical practice could revolutionize early detection and risk stratification, ultimately improving the management of this aggressive cancer. Future studies should focus on validating these findings and exploring survivin’s potential as a therapeutic target. Our study reinforces survivin’s role as a high-performance biomarker for GBC, with diagnostic and prognostic metrics matching or exceeding prior reports. While CEA and CA19-9 remain useful, their limitations underscore the need for novel markers like survivin. Future research should focus on multicenter validation, survivin-targeted therapies, and integrated biomarker panels to optimize GBC management.

1. Hundal EA, Shaffer EA. Gallbladder cancer: epidemiology and outcome. Clin Epidemiol. 2014;6:99-109.
2. Wistuba II, Gazdar AF. Gallbladder cancer: lessons from a rare tumour. Nat Rev Cancer. 2004;4:695-706.
3. Huang J, Patel HK, Boakye D, Chandrasekar VT, Koulaouzidis A, Lucero-Prisno DE 3rd, et al. Worldwide distribution, associated factors, and trends of gallbladder cancer: a global country-level analysis. Cancer Lett. 2021;521:238-51. doi:10.1016/j.canlet.2021.09.004.
4. Dwivedi AN, Jain S, Dixit R. Gall bladder carcinoma: Aggressive malignancy with protean loco-regional and distant spread. World J Clin Cases. 2015;3(3):231-44. doi: 10.12998/wjcc.v3.i3.231. PMID: 25789296; PMCID: PMC4360495.
5. He JJ, Xiong WL, Sun WQ, Pan QY, Xie LT, Jiang TA. Advances and current research status of early diagnosis for gallbladder cancer. Hepatobiliary Pancreat Dis Int. 2024. doi: 10.1016/j.hbpd.2024.09.011. Epub ahead of print. PMID: 39393997.
6. Andrén-Sandberg A. Diagnosis and management of gallbladder cancer. N Am J Med Sci. 2012;4(7):293-9. doi: 10.4103/1947-2714.98586. PMID: 22866265; PMCID: PMC3409652.
7. Wen Z, Si A, Yang J, Yang P, Yang X, Liu H, et al. Elevation of CA19-9 and CEA is associated with a poor prognosis in patients with resectable gallbladder carcinoma. HPB (Oxford). 2017;19(11):951-6. doi:10.1016/j.hpb.2017.06.011.
8. Goonetilleke KS, Siriwardena AK. Systematic review of carbohydrate antigen (CA 19-9) as a biochemical marker in the diagnosis of pancreatic cancer. Eur J Surg Oncol. 2007;33:266-70.
9. Hatzaras I, Schmidt C, Muscarella P, Melvin WS, Ellison EC, Bloomston M, et al. Elevated CA 19-9 portends poor prognosis in patients undergoing resection of biliary malignancies. HPB. 2010;12:134-38.
10. Grunnet M, Mau-Sørensen M. Serum tumor markers in bile duct cancer: A review. Biomarkers. 2014;19:437-43.
11. Zhang Y, Yang J, Li H, Wu Y, Zhang H, Chen W, et al. Tumor markers CA19-9, CA242 and CEA in the diagnosis of pancreatic cancer: a meta-analysis. Int J Clin Exp Med. 2015;8:11683-91.
12. Reitz D, Gerger A, Seidel J, Kornprat P, Samonigg H, Stotz M, et al. Combination of tumour markers CEA and CA19-9 improves the prognostic prediction in patients with pancreatic cancer. J Clin Pathol. 2015;68:427-33.
13. Nigam J, Chandra A, Kazmi HR, Parmar D, Singh D, Gupta V. Prognostic significance of survivin in resected gallbladder cancer. J Surg Res. 2015;194(1):57-62. doi:10.1016/j.jss.2014.07.054.
14. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th ed. of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 2010;17:1471-1474.
15. Yang XW, Yuan JM, Chen JY, Yang J, Gao QG, Yan XZ, et al. The prognostic importance of jaundice in surgical resection with curative intent for gallbladder cancer. BMC Cancer. 2014;14:652.
16. Nigam J, Chandra A, Kazmi HR, Singh A, Parmar D, Srivastava MK, et al. Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: a comparative study. Med Oncol. 2014;31:167. doi:10.1007/s12032-014-0167-5.
17. Aloia TA, Járufe N, Javle M, Maithel SK, Roa JC, Adsay V, et al. Gallbladder cancer: expert consensus statement. HPB. 2015;17:681-690.
18. Nigam J, Chandra A, Kazmi HR, Singh A, Gupta V, Parmar D, et al. Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: a comparative study. Med Oncol. 2014;31(167). doi:10.1007/s12032-014-0167-5.
19. Gupta V, Goel MM, Chandra A, Gupta P, Kumar S, Nigam J. Expression and clinicopathological significance of antiapoptotis protein survivin in gallbladder cancer. Indian Journal of Pathology and Microbiology. 2016 Apr 1;59(2):143-7.
20. Salman T, Argon A, Kebat T, et al. The prognostic significance of survivin expression in gallbladder carcinoma. APMIS. 2016;124(8):633–638. DOI: https://doi.org/10.1111/apm.12551..
21. Strom BL, Maislin G, West SL, Atkinson B, Herlyn M, Saul S, Rodriguez‐Martinez HA, Rios‐Dalenz J, Iliopoulos D, Soloway RD. Serum CEA and CA 19‐9: potential future diagnostic or screening tests for gallbladder cancer?. International journal of cancer. 1990 May 15;45(5):821-4.
22. Sachan A, Saluja SS, Nekarakanti PK, Nimisha, Mahajan B, Nag HH, Mishra PK. Raised CA19-9 and CEA have prognostic relevance in gallbladder carcinoma. BMC Cancer. 2020 Aug 31;20(1):826. doi: 10.1186/s12885-020-07334-x. PMID: 32867709; PMCID: PMC7457344.
23. Wen Z, Si A, Yang J, Yang P, Yang X, Liu H, Yan X, Li W, Zhang B. Elevation of CA19-9 and CEA is associated with a poor prognosis in patients with resectable gallbladder carcinoma. Hpb. 2017 Nov 1;19(11):951-6.
24. Kim M, Kim H, Han Y, Sohn H, Kang JS, Kwon W, Jang JY. Prognostic value of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) in gallbladder cancer; 65 IU/ml of CA 19-9 is the new cut-off value for prognosis. Cancers. 2021 Mar 4;13(5):1089.
25. Yu T, Yu H, Cai X. Preoperative prediction of survival in resectable gallbladder cancer by a combined utilization of CA 19–9 and carcinoembryonic antigen. Chinese medical journal. 2014 Jun 20;127(12):2299-303.
26. Liu C, Sun B, An N, et al. Inhibitory effect of Survivin promoter-regulated oncolytic adenovirus carrying P53 gene against gallbladder cancer. Mol Oncol. 2011;5(6):545-554. DOI: https://doi.org/10.1016/j.molonc.2011.10.001