Abstract
Gallbladder carcinoma (GBC) is more frequent after 60 years of age; its behavior in young adults has not been much studied. A retrospective analysis was performed in patients who underwent a cholecystectomy procedure between the years 2001 to 2016. A group of young patients (< 45) were compared with elderly patients (> 60 years) with reference to various clinical, histomorphologic, and immunohistochemical parameters. Statistical analysis was performed using t test and Fisher’s test. Survival curves were calculated by Kaplan-Meier actuarial survival curves and log-rank tests. One hundred and one patients with GBC were observed during the study period. Of these, 14 patients (13.9%) belonged to the study group (age range 20 to 45 years) and 43 patients (42.6%) constituted the comparison elderly control group (age range 60 to 80 years). Forty-four pts. were in the middle-aged group (46 to 59 years) and were thus excluded from the study. With reference to age (< 45 and > 60), no significant difference was found in sex (females 64.3% vs 69.8%, p = 0.7), presence of gall stones (64% vs 60%, p = 0.8), advanced disease at presentation (T4) (14.3% vs 7%, p = 0.40), incidental detection of gallbladder carcinoma (28.5% vs 28%, p = 0.9), tumor stage at presentation (stage I/II) (35.7% vs 49%, p = 0.39), and poor differentiation (tumor grades G3) (14% vs 12%, p = 0.79). Full-length involvement (28.5% vs 11.6%, p = 0.015) of the gallbladder and abundant tumor necrosis (43% vs 14%, p = 0.021) were more common in the younger patients group whereas adenosquamous and pure squamous cell carcinoma were predominantly observed in elderly patients. Immunohistochemical studies showed higher percentage of overexpression of p53 and Ki-67 proliferation indices in the younger population. Overall survival in younger patients was 48 months whereas in elderly patients it was 36 months. Histological markers denoting aggressive tumor behavior were observed in gallbladder carcinomas of younger individuals; further studies are needed to delineate the differences in molecular mechanisms involved in progression of the tumor in the two groups.
Keywords: Gallbladder carcinoma, Young adults, Elderly
Introduction
The incidence of gallbladder carcinoma (GBC) has a worldwide variation as well as in the Indian subcontinent. The highest risk of cancer is observed in northeast and central regions of India and is lowest in South India. [1] Females are more prone to develop GBC compared with males and studies have shown that the risk varies 2 to 4 times. [2] A close association between GBC and gall stones has been demonstrated while other risk factors for GBC mentioned in various studies include old age, female sex, obesity, dietary pattern, environmental factors, genetic factors, female reproductive factors, and chronic inflammation. [3–5]
A lack of recognizable premalignant state precludes screening leading to recognition of malignancy in advanced stages in a significant proportion of patients. [6] Trend analysis has revealed a recent increase in the incidence of late stage GBC in USA also. [7] Among the emerging cancer trends in the young population, the incidence for obesity-related cancers (multiple myeloma, colorectal, uterine corpus, gallbladder, kidney, and pancreatic) has increased significantly. [8] The incidence of GBC increases after the age of 45 years and is maximum at the age of 65 years. A significant upward trend for GBC has been observed in young individuals in the past decades. [9]
The behavior of GBC in young individuals (< 45 years) has not been studied much with only a few studies present in medical literature worldwide. The objective of the present study was to assess the relationship between age and clinicopathological characteristics of GBC.
Material and Methods
Patients with GBC admitted from January 2001 to December 2016 that had clinical or radiological suspicion of malignancy and underwent cholecystectomy at our institute were analyzed. Incidentally detected malignancies on histological examination were also included in the study. Detailed data were collected from patients’ case records. Two patient groups were formed wherein young patients (< 45 years) were compared with the elderly patients (> 60 years) for various clinicopathological parameters. Clinical variables studied were gender, presenting signs and symptoms, clinical examination, presence of gall stones, type of surgery, and incidental detection of malignancy.
The pathological parameters studied included maximum tumor size, site of the tumor, tumor dimensions (> 2 cm), type of growth, histologic subtype of carcinoma, grade of tumor differentiation, perineural and/or perivascular invasion, lymph node involvement, and liver involvement. Necrosis in the tumor was scored as focal (10%), intermediate (11–25%), and abundant (> 25%). The histological changes in adjacent gallbladder epithelium were also studied in detail for various types of metaplasia, xanthogranulomatous inflammation, and dysplasia. The histomorphologic parameters were individually assessed by three independent pathologists. The study was ethically approved by the institutional ethical committee.
Immunohistochemical stains were carried out using 3-μm-thick sections on poly-l-lysine–coated slides by standard horseradish peroxidase technique. Antibodies against HER2neu, p53, and Ki-67 were performed on each case in both groups. HER2neu was scored from 0 to 3: score 2 was taken as complete membrane staining in 10–30% of cells, whereas score 3 was regarded as > 30% complete membrane positivity in tumor cells. p53 positivity was established when > 50% of tumor cells exhibited nuclear staining. Ki67 proliferation index was calculated by the manual counting method wherein an initial slide scan was performed to identify the tumor “hot spots” and the × 10 objective was then used to count the Ki67-positive cells per 2000 tumor cells in real time (< 10%, low; 10–20%, intermediate; > 20%, high). Staging and grading of tumor were done in accordance with American Joint Cancer Committee (AJCC) 2016 for the tumor-node-metastasis (TNM) classification. Deaths due to unrelated causes were considered to have died from GBC.
Statistical Analysis
For statistical analysis, data were entered into a Microsoft Excel spreadsheet and then analyzed by Statistical Package for Social Sciences 20.0.1 and GraphPad Prism version 5. Data was summarized as mean and standard deviation for numerical variables and count and percentages for categorical variables. Student’s independent samples t test was applied to compare normally distributed numerical variables between groups and unpaired proportions were compared by chi-square test or Fisher’s exact test as appropriate. Kaplan-Meier estimator (Kaplan-Meier survival analysis) was a nonparametric statistic test used to estimate the survival function from time data. Cumulative survival rates and survival curves were calculated by Kaplan-Meier actuarial survival curves and log-rank tests. The Cox proportional hazards model was used to identify the hazard ratio (HR) and 95% confidence interval (CI) of the clinical outcomes along with significant covariates for negative and positive groups. p value < 0.05 was considered statistically significant.
Results
During the study period, a total of 101 patients (n = 101) with GBC were identified. The young patient group (< 45 years) accounted for 14% (n = 14) patients and the elderly group constituted (> 60 years) 43% (n = 43) of total patients. Forty-four pts. were in the middle-aged group (46 to 59 years) and were thus excluded from the study. The mean age of presentation in the young group (n = 14) was 37 years (median 40 years, range 20–45 years). Female predominance was observed in both groups. Demographic data with clinical parameters have been summarized in Table 1. With respect to age (< 45 and > 60 years), no statistically significant difference was observed in presenting signs and symptoms, presence of gall stones, tumor size, location of tumor in gallbladder, and tumor growth pattern. Histologically, adenocarcinoma was the commonest subtype in both the young and elderly individuals. Unusual malignancies such as carcinosarcoma, neuroendocrine differentiation, and clear cell morphology were observed in one case each in the young patient group (Figs. 1, 2, 3). On the contrary, adenosquamous and pure squamous cell carcinoma were almost exclusively observed in the elderly age group. With reference to the other histomorphological parameters studied, younger patients (< 45 years) were found to have more frequent involvement of full length of gallbladder by malignancy, greater likelihood of perineural invasion, and presence of abundant necrosis (> 25%). Morphological changes studied in the rest of the gallbladder also revealed no statistically significant variations (refer to Table 2).
Table 1.
Demographic profile and clinical characteristics of gallbladder carcinoma patients
| S. no. | Clinical parameters | Young pt (n = 14) |
Elderly pt (n = 43) |
p value |
|---|---|---|---|---|
| 1. | Sex ratio (male:female) | 5:9 | 13:30 | 0.7 |
| 2. | Age range (years) | 20–45 years | 60–80 years | – |
| 3. | Commonest decade | 30–40 years | 60–70 years | – |
| 4. | Type of surgery | |||
| Cholecystectomy (full/partial) | 4 (28.5%) | 15 (35%) | ||
|
Radical procedure (liver wedge+LN *) |
10 (71.5%) | 19 (44%) | ||
|
Extended resection (stomach/colon/duodenum) |
– | 9 (21%) | ||
| 5. | Presenting signs and symptoms | |||
| Pain | 9 (64%) | 33 (76%) | 0.35 | |
| Fever | 5(35%) | 9(20%) | 0.2 | |
| Vomiting | 6(43%) | 12(28%) | 0.3 | |
| Jaundice | 5(35%) | 9(20%) | 0.26 | |
| 6. | Incidental malignancy | 4 (28.5%) | 12 (28%) | 0.9 |
| 7. | Gall stones | 9 (64%) | 26 (60%) | 0.8 |
| 8. | Maximum tumor size (range) | 1.5–20 cm | 0.5–8.5 cm | – |
| 9. | Tumor size >2 cm | 12 (86%) | 35 (81%) | 0.7 |
| 10. | Tumor site | |||
| Fundus | 4 (29%) | 17 (40%) | 0.46 | |
| Body | - | 5 (12%) | ||
| Neck | 1 (7%) | - | ||
| Fundus + body | 3 (21%) | 15 (35%) | ||
| Body + neck | 2 (14%) | 1 (2%) | ||
|
Fundus + body + neck (full length) |
4 (29%) | 5 (11%) | 0.015 | |
| 11. | Growth type | |||
| Ulceroproliferative | 8 (57%) | 26 (60%) | ||
| Ulceroinfiltrative | 5 (36%) | 14 (33%) | NS | |
| Both | 1 (7%) | 3 (7%) | ||
LN, lymph node
Fig. 1.
Histologic grades of gallbladder carcinoma. a Grade 1: well-differentiated adenocarcinoma. b Grade 2: moderately differentiated adenocarcinoma with presence of glands as well as groups of tumor cells invading the muscle layer. c Grade 3: poorly differentiated adenocarcinoma showing poorly cohesive tumor cells
Fig. 2.
a Perineural invasion by tumor cells. b Vessel wall infiltration in a case of gallbladder adenocarcinoma. c Extensive tumor necrosis in a case of poorly differentiated adenocarcinoma with few viable tumor cells
Fig. 3.
Immunohistochemical marker expression in GBC. a HER2neu showing strong complete membranous staining in 45% of tumor cells (3+ positivity). b p53 overexpression observed in 70 to 80% of tumor cells. c Ki-67 proliferation marker exhibits nuclear positivity in ~ 70% of tumor cells
Table 2.
Histomorphological characteristics in gallbladder carcinoma patients
| Histomorphological features | Young pt (n = 14) |
Elderly pt (n = 43) |
p value | |
|---|---|---|---|---|
| 1. | Histological subtype | |||
| Adenocarcinoma | 13 (93%) | 40 (93%) | NS | |
| Adenosquamous and pure SCC | – | 3 (7%) | ||
| Others (carcinosarcoma) | 1 | – | ||
| 2. | Histologic variants | |||
| Papilla formation | 5 (36%) | 14 (32.5%) | NS | |
| Intestinal type | 1 | 3 | ||
| Mucinous pools (+ signet ring) | 3 | 3 | ||
| Clear cell | 1 | 2 | ||
| Neuroendocrine | 1 | – | ||
| 3. | Grade of differentiation | |||
| G1 (well > 95%) | 1 (7%) | 10 (23%) | 0.18 | |
| G2 (moderate 50–95%) | 11 (79%) | 28 (65%) | 0.34 | |
| G3 (poor < 49%) | 2 (14%) | 5 (12%) | 0.79 | |
| 4. | Perineural + perivascular Inv | 11 (79%) | 18 (42%) | 0.017 |
| 5. | Liver involvement | 9 (64%) | 15 (35%) | 0.05 |
| 6. | Lymph node involvement | 4 (28.5%) | 17 (39.5%) | 0.46 |
| 7. | Necrosis: focal (0–10%) | 5 (36%) | 22 (51%) | 0.31 |
| Intermediate (11–25%) | 3(21%) | 15(35%) | 0.34 | |
| Abundant (> 25%) | 6 (43%) | 6(14%) | 0.021 | |
| 8. | Gallbladder dysplasia (adjacent epithelium) | 9 (64%) | 24 (56%) | 0.57 |
| 9. | Metaplasia | |||
| Pyloric gland | 4 (28%) | 16 (37%) | 0.55 | |
| Intestinal | 6 (14.8%) | 17 (39%) | 0.88 | |
| 10. | Xanthogranulomatous inflammation | 1 (7%) | 6 (14%) | 0.5 |
| 11. | Inflammatory cells | |||
| Lymphocytes | 13 (93%) | 40 (93%) | 0.9 | |
| Neutrophils | 10(71.4%) | 37 (86%) | 0.4 | |
| Eosinophils |
9 (64.2%) 9 (64.2%) |
19 (44.1%) 21 (49%) |
0.19 0.31 |
|
| Histiocytes | ||||
The immunohistochemical markers performed in both the groups also showed similar scores except for higher Ki-67 proliferation index (> 20%) in 93% of the younger group in comparison with 51% patients of the elderly group (p value 0.005) (refer to Table 3). Although the overall survival and 5-year survival rate were statistically insignificant between the two groups, the younger patients had better mean survival (48 months vs 36 months). The total follow-up duration for the patients ranged from 10 days to 144 months (1–120 months in younger vs 10 days to 144 months in elderly group) (refer to Table 4).
Table 3.
Immunohistochemical expression of markers in gallbladder carcinoma patients
| Immunohistochemistry | Young pt (n = 14) |
Elderly pt (n = 43) |
p value | |
|---|---|---|---|---|
| 1. | HER2neu | |||
| Score 0 and 1 | 11 (79%) | 36 (84%) | 0.65 | |
| Score 2 | 2 (14%) | 3 (7%) | 0.40 | |
| Score 3 | 1 (7%) | 4 (9%) | 0.23 | |
| 2. | p53 | |||
| Strong/weak | ||||
| > 50% | 8 (57%) | 20 (47%) | 0.49 | |
| < 50% | 6 (43%) | 23 (53%) | 0.48 | |
| 3. | Ki-67 index | |||
| Low (< 10%) | – | 9 (21%) | ||
| Intermediate (10–20%) | 1 (7%) | 12 (28%) | ||
| High (> 20%) | 13 (93%) | 22 (51%) | 0.005 | |
Table 4.
Comparative data for stage, clinical follow-up, and survival of patients in both groups
| Stage and survival | Young (n = 14) | Elderly (n = 43) |
p value | |
|---|---|---|---|---|
| 1. | Primary tumor (pT) | |||
| pT1 | 1 (7%) | 8 (19%) | 0.30 | |
| pT2 | 4 (29%) | 21 (49%) | 0.18 | |
| pT3 | 7 (50%) | 11 (25%) | 0.08 | |
| pT4 | 2 (14%) | 3 (7%) | 0.40 | |
| 2. | Tumor stage | |||
| Stage 1 | 1 (7%) | 8 (19%) | 0.30 | |
| Stage 2 | 4 (29%) | 13 (30%) | 0.90 | |
| Stage 3 | 7 (50%) | 19 (44%) | 0.70 | |
| Stage 4 | 2 (14%) | 03 (7%) | 0.40 | |
| 3. | 3-year survival | 7 (50%) | 9 (21%) | 0.035 |
| 5-year survival | 4 (29%) | 7 (16%) | 0.31 | |
| 4. | Follow-up in months (range) | 1–120 months | 10 days–144 months | – |
Independent predictors for overall survival were lymph node involvement, perineural and/or perivascular invasion, and TNM stage of the tumor. Presence of abundant tumor necrosis, higher proliferation index, or overexpression of p53 did not have significant correlation with overall survival in GBC patients. Table 5 displays the results of Cox proportional hazards survival model (Fig. 4, 5, 6).
Table 5.
Adjusted Cox proportional hazards regression model for overall survival in patients with gallbladder carcinoma
| Variables | n | Overall survival (months) |
Univariate analysis | |
|---|---|---|---|---|
| HR (95% CI) | p | |||
| Age | ||||
| < 45 years | 14 | 48.19 | 26.14–70.23 | 0.308 |
| > 60 years | 43 | 36.46 | 20.32–52.59 | |
| Full-length GBC | ||||
| Yes | 9 | 46.14 | 15.42–77.38 | 0.276 |
| No | 48 | 36.38 | 23.32–49.86 | |
| Grade | ||||
| High G3 | 6 | 12.34 | 5.80–19.61 | 0.166 |
| Low G1/G2 | 51 | 44.63 | 29.76–59.51 | |
| Lymph node inv | ||||
| Yes | 21 | 12.71 | 6.90–18.52 | 0.0001 |
| No | 36 | 61.51 | 41.12–81.91 | |
| Perineural Inv | ||||
| Yes | 29 | 25.92 | 13.5–38.34 | 0.041 |
| No | 28 | 57.91 | 34.63–81.19 | |
| Abundant necrosis (> 25%) | ||||
| Yes | 17 | 26.08 | 11.08–41.07 | 0.175 |
| No | 40 | 46.68 | 28.79–64.57 | |
| Ki-67 (> 20%) | ||||
| Present | 40 | 32.21 | 20.38–44.05 | 0.176 |
| Absent | 17 | 62.35 | 28.54–96.14 | |
| p53 (> 50%) | ||||
| Present | 28 | 33 | 19.56–48.21 | 0.792 |
| Absent | 29 | 44 | 24.67–65.32 | |
| TNM stage | ||||
| I–II | 25 | 66 | 39.54–92.31 | 0.0056 |
| III–IV | 32 | 25 | 13.46–37.23 | |
HR, hazard ratio; 95% CI, 95% confidence interval; GBC, gallbladder cancer; TNM, tumor-node-metastasis
Fig. 4.
Kaplan-Meier plot for overall survival in young and elderly patients with gallbladder carcinoma
Fig. 5.
Correlation analysis between high proliferation index (Ki-67 > 20%) and overall survival
Fig. 6.
Kaplan-Meier analysis showing survival distribution curves in the various (TNM) stages of gallbladder carcinoma
Discussion
Gallbladder carcinoma is predominantly a disease of the elderly and is uncommon in younger individuals (< 45 years of age). [10] It has been observed that GBC shows marked geographical and racial variations in the incidence of malignancy worldwide. Even within India, there is regional diversity with a majority of the cases being reported from northern (Delhi) and the central part of country, i.e., Bhopal. [11] In the present study, younger patients constituted 14% of gallbladder malignancies. In a Delhi population-based cancer registry, among 2402 newly diagnosed cases of GBC diagnosed between the years 2001–2005, 20% of them belonged to the age group below 45 years. [12] As per the data of Delhi cancer registry, a significant upward trend in incidence of gallbladder carcinoma was observed in younger individuals. [6] Female predominance was observed in both young (1.8 times) and elderly (2.3 times) age groups in our study. As per the 25-year trend analysis of gallbladder carcinoma in urban Delhi population by Malhotra RK et al., there was a significant upward trend in both males and females between 20 and 39 age groups. The 40–49 age group revealed an increasing trend of GBC after 2008 in males, whereas in females there was a linear increasing trend. [9] In a prospective observational study of GBC in eastern India, females were affected 2.4 times as males. Three-fourths of their cases had associated cholelithiasis; patients with multiple stones less than 3 cm were found to have greater risk of malignancy. [13] Incidence of gallstones in association with malignancy in the present study was nearly 60%.
Routine histopathological examination of the gallbladder is significant as incidental detection of gallbladder cancer may occur. [14, 15] In a study of gallbladder cancer in young Korean patients, distinct clinicopathological features such as increased frequency of carcinoma arising in adenoma and rare association of intestinal metaplasia were observed. [16] However, in the present study, no such differences were noted; the presence of gall stones, intestinal metaplasia, and dysplasia in the adjacent gallbladder epithelium were almost similar in both groups. In contrast, features of aggressiveness such as abundant tumor necrosis (> 25%) and high Ki-67 proliferation index (> 20%) were statistically significant in younger individuals. The aggressive behavior of gallbladder malignancy in younger patients (< 45 years) has also been reported by authors Kazmi HR et al. [17] Age-related differential expression of cholecystokinin A receptor was also observed in gallbladder cancer in young and elderly individuals. [17]
In addition, the present study highlighted the fact that full-length involvement of gallbladder by malignancy was more frequent in younger patients (28.5% vs 11%; p = 0.015) and perineural invasion was also more frequent in them (79% vs 42%; p = 0.017). Similar to the observations in medical literature, adenocarcinomas were the most common subtype in both groups; pure squamous cell carcinoma and adenosquamous gallbladder carcinoma were predominantly reported in the elderly individuals and were a rarity in younger individuals. [18, 19]
Tumor suppressor gene (p53) is lost or mutated in half of all the human cancers. [20] Although p53 overexpression was observed in 57% of patients, its positivity or even high proliferation index (Ki-67 index > 20%) did not directly correlate with the TNM stage or patient survival. Similar findings were observed by Grau et al. in an immunohistochemical study of 41 cases of gallbladder carcinoma. [21] According to another study, patients with high Ki-67 index significantly correlated with lymphatic and vascular invasion by tumor cells and concluded that high Ki-67 index predicts early recurrence after surgery for gallbladder carcinoma. [22]
Her-2-neu overexpression was observed in approximately 15–20% of GBCs in the present study. Drug trials are still under evaluation for utilizing anti-Her-2 therapy in GB malignancy. [23] Other studies have demonstrated the fact that target population with Her-2 overexpression may consist of only about 12% of all GBCs, making the targeted therapy even more challenging. [24] However, the interest in Her-2-neu signaling pathway and its role in GB carcinogenesis may yield promising clinically actionable target for this rare orphan gastrointestinal cancer. [25]
With reference to overall survival analysis, the mean survival showed an estimate of stage-wise comparison and overall survival. Only the resectable gallbladder malignancies were included in the present study, and the survival rates for stage I, stage II, stage III, and stage IV were 85.5, 55, 22, and 20 months, respectively (overall survival 40.6 months). As expected, the younger patients had better mean survival of 48 months vs 36 months (elderly group). In a study by Gupta et al., median survival was 38 months in both young and elderly GBC patients with no significant differences in survival curves. Unresectability due to local infiltration was significantly lower in young patients. [26] In a clinical evaluation of the survival rate for gallbladder carcinoma at a tertiary center, the survival rates for stage II, stage IIIa, stage IIIb, stage IVa, and stage IVb were > 12, 10, 8.75, 4.5, and 1.5 months, respectively. [27] Stage at presentation and ability to perform curative resection were the most important prognostic factors predicting survival. [28]
To conclude, although there was not much difference in clinicopathological parameters among the two groups, the tumors in young individuals exhibited certain features of histological aggression in the tumor cells. Genome-wide analysis and other molecular genetic studies are needed in the future to better understand the pathogenetic mechanisms involved in gallbladder carcinogenesis and tumor progression. HER2neu overexpression, an attractive target for treatment, is still in evolution phase. Clinical drug trials in combination with anti-HER2neu therapy may further improve survival in this selected group of patients.
Acknowledgments
Histopathology staff (Mrs Kahkasha Parveen and peer colleagues) at BMHRC. Dr. Yogesh Sabde, Scientist E, NIREH, for his critical support in statistical analysis.
Author Contributions
Concepts: Gupta Suneeta, Gulwani Hanni, Kaur Sukhpreet
Design: Gulwani Hanni, Kaur Sukhpreet
Definition of intellectual content: Gulwani Hanni
Literature search: Gupta Suneeta, Gulwani Hanni, Kaur Sukhpreet
Clinical studies: Gupta Suneeta, Gulwani Hanni, Kaur Sukhpreet
Experimental studies: Gulwani Hanni
Data acquisition: Gupta Suneeta, Gulwani Hanni
Data analysis: Gupta Suneeta, Gulwani Hanni
Statistical analysis: Gupta Suneeta, Gulwani Hanni
Manuscript preparation: Gupta Suneeta, Gulwani Hanni
Manuscript editing: Gupta Suneeta, Gulwani Hanni, Kaur Sukhpreet
Manuscript review: Gupta Suneeta, Gulwani Hanni, Kaur Sukhpreet
Guarantor: Gupta Suneeta, Gulwani Hanni, Kaur Sukhpreet
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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