- Email: [email protected]
Neuroendocrine differentiation in extrahepatic bile duct carcinomas and its prognostic significance
Human Pathology (2005) 36, 732 – 740
www.elsevier.com/locate/humpath
Neuroendocrine differentiation in extrahepatic bile duct carcinomas and its prognostic significance Seung-Mo Hong MD, PhD, Mi-Jung Kim MD, David Y. Pi, Daniel Jo, Eunsil Yu MD, PhD, Jae Y. Ro MD, PhD*,1 Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, South Korea Received 21 December 2004; accepted 3 May 2005
Keywords: Extrahepatic bile duct carcinoma; Immunohistochemistry; Neuroendocrine differentiation; Synaptophysin; Chromogranin
Summary Neuroendocrine differentiation is known to be one of the prognostic factors in many carcinomas. However, the characteristics of neuroendocrine differentiation are not well elucidated in extrahepatic bile duct (EBD) carcinomas. One hundred ninety-four cases of EBD carcinomas were analyzed using immunohistochemistry with synaptophysin and chromogranin. The tumors were graded as degree 0, 1, and 2 when the positive tumor cells were 5% or less, 6% to 25%, and 26% or more, respectively. Immunohistochemical results were compared with clinicopathologic variables and survival rate. Synaptophysin and chromogranin were positive in 54 (27.8%) and 74 (38.1%) cases, respectively. Thirty-four cases (17.5%) were positive for both synaptophysin and chromogranin, 20 (10.3%) and 40 cases (20.6%) were positive only for synaptophysin and for chromogranin, respectively, and 100 cases (51.6%) were negative for both markers. There was a significant survival difference between overall synaptophysin-positive (median, 27 months) and synaptophysin-negative (38 months) groups ( P b .05). However, there was no survival difference between chromograninpositive and chromogranin-negative groups. There was a significant survival difference between the dual-positive expression to synaptophysin and chromogranin group (median, 21 months) and the dualnegative expression group (median, 35 months; P b .05). In summary, synaptophysin expression was an important prognostic factor because synaptophysin-positive cases showed a worse prognosis than synaptophysin-negative cases. The more tumor cells expressed chromogranin, the poorer the survival. Therefore, immunohistochemical studies for neuroendocrine differentiation may be helpful in routine pathological examinations for evaluating the survival and the prognosis of patients with EBD carcinomas. D 2005 Elsevier Inc. All rights reserved.
1. Introduction
T Corresponding author. E-mail addresses: [email protected], [email protected] (J.Y. Ro). 1 Current address: Department of Pathology, The Methodist Hospital, 6565 Fannin Street, Houston, TX 77030. 0046-8177/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2005.05.002
Extrahepatic bile duct (EBD) carcinoma is an uncommon neoplasm with a dismal prognosis. Two- to 5-year median survival of localized EBD carcinoma was reported to be about 20% to 40% [1]. Several factors have been demonstrated as prognostic factors, such as histological type,
Neuroendocrine differentiation histological grade, site of tumor, depth of invasion, duodenal and pancreatic invasion, involvement of the resection margin, and vascular, lymphatic, and perineural invasion [1-8]. Neuroendocrine differentiation has been reported in neoplasms from many different organs with a variable prognostic significance. It is reported to be one of the worst prognostic factors in prostate [9-11], kidney [12], thyroid gland [13], lung [14,15], and colon [16,17] carcinomas. On the other hand, the presence of neuroendocrine differentiation showed a better survival in pancreatic carcinoma [18], and no prognostic significance in other neoplasms such as breast [19], stomach [20,21], esophagus [22], and cervix [23] carcinomas. Although a few small series have been reported, neuroendocrine differentiation in EBD carcinomas is not well characterized [24,25], and clinicopathologic significance of the neuroendocrine differentiation is still uncertain. A previous study from a Western country reported that neuroendocrine differentiation was associated with a shorter survival rate [24]; however, this study is not fully accepted because of the limited number of cases. Therefore, an additional clinicopathologic study on neuroendocrine differentiation in EBD carcinomas with a large number of cases is required to validate the issue. In this study, we analyzed neuroendocrine differentiation in a large number of EBD carcinomas and compared the results with clinicopathologic parameters to clarify whether neuroendocrine differentiation has a prognostic impact to the patients.
2. Materials and methods 2.1. Case selection One hundred ninety-four surgically resected cases with available tissue blocks for immunostaining of EBD carcinoma from the files of the Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea, from 1991 to 2000, were included in this study. Only carcinomas with the epicenter in the EBD were included in this study. Either carcinomas with the epicenter in the ampulla of Vater or pancreas or carcinomas with obvious precancerous epithelial changes in the ampulla of Vater or pancreas were excluded. In addition, equivocal cases in terms of origin of the tumors were also excluded from this study. Carcinomas arising from the gallbladder and intrahepatic bile duct with extension to the EBD were also excluded from this study. Data obtained from reviewing medical records included patient’s age, sex, survival time, and survival status, and from pathology reports were tumor location, size, and growth pattern. Information on postoperative radiation and/or chemotherapy and performance status of patients were not analyzed in this study.
2.2. Pathological study Microscopic investigation was made on the hematoxylineosin–stained slides. Histological type, depth of tumor
733 invasion, metastasis to lymph nodes, vascular and perineural invasion, involvement of the resection margin, and involvement of the pancreas, duodenum, and liver were evaluated.
2.3. Immunohistochemical staining A representative paraffin-embedded tissue block from each case was selected for immunohistochemical study. Immunohistochemical staining was carried out by the streptoavidin-biotin method, using a commercially available kit. Deparaffinized sections were treated with methanol containing 3% H2O2 for 10 minutes after 2 times antigen retrieval using a microwave processor at 958C for 5 minutes. After washing with phosphate-buffered saline, blocking serum was applied for 10 minutes. Then, primary antibodies, synaptophysin (antimouse synaptophysin monoclonal antibody, Dinnona, Inc, Seoul, South Korea; 1:200 dilution), and chromogranin (antimouse chromogranin monoclonal antibody, Dinnona; 1:200 dilution) were allowed to react at room temperature for 1 hour. After washing in phosphate-buffered saline, biotin-marked secondary antibody was applied for 10 minutes followed by peroxidase-marked streptoavidin for 10 minutes on both antibodies. The reaction was visualized by diaminobenzidine tetrahydrochloride. Nuclei were stained with Harris hematoxylin. Sections from normal pancreas containing islet cells were used as positive controls for synaptophysin and chromogranin. Negative controls were performed by substituting a nonimmune purified mouse monoclonal antibody for the primary antibodies.
2.4. Interpretation of immunohistochemical staining The immunoreactivity for each case was interpreted as positive and negative, and then graded as degree 0, 1, and 2 when 5% or less, 6% to 25%, and 26% or more, respectively, of the tumor cells were positive for synaptophysin and chromogranin (Fig. 1). We counted positive cells among 500 cells in the most active areas and provided actual numbers of positive percentage.
2.5. Comparison of neuroendocrine differentiation with clinicopathologic parameters Immunohistochemical results of the synaptophysin and chromogranin were compared with clinicopathologic variables, which included age, sex, location, size, growth patterns, histological types, T classification, marginal status, nodal metastasis, survival rate, and vascular, perineural, pancreatic, hepatic, and duodenal invasion. The presence of neuroendocrine differentiation was defined as any or both neuroendocrine markers positivity and was compared with the abovementioned clinicopathologic variables. Expression to both synaptophysin and chromogranin, to synaptophysin only, and to chromogranin only and no expression to either synaptophysin or chromogranin
734
S.-M. Hong et al. Prognostic factors were tested by univariate and multivariate analyses (proportional hazards regression model). P b .05 was considered statistically significant.
3. Results 3.1. Clinicopathologic characteristics of cases The ages of the patients ranged from 30 to 84 years (mean, 60 years; SD, 10 years). One hundred thirty-nine cases were men and 55 were women. The tumors showed infiltrative growth pattern in 152 cases, papillary in 25, and nodular in 17 cases. The tumor size was less than 2 cm in 83 cases, between 2 and 4 cm in 93 cases, and more than 4 cm in 18 cases. There were 98 proximal (perihilar) tumors, 88 distal, and 8 diffuse tumors. Forty cases were confined within the bile duct (T1 classification), 65 cases invaded the fibroadipose tissue (T2 classification), 79 were T3 tumors, and 10 were T4 tumors. In addition, there were 10 cases of hepatic invasions, 78 pancreatic invasions, 129 perineural invasions, and 47 vascular invasions. Tumor cells involved the resection margins in 44 cases, whereas 62 cases showed metastasis to lymph nodes. Histological
Fig. 1 Degree of the immunohistochemical staining. A, No tumor cells were stained with synaptophysin (degree 0, 200). B, Ten percent of tumor cells were positive for synaptophysin (degree 1, 200). C, Seventy percent of tumor cells were positive for synaptophysin (degree 2, 200).
were also compared with the abovementioned clinicopathologic variables. After that, coexpression versus no expression to both markers was compared with the clinicopathologic variables.
2.6. Statistical analyses Statistical analyses were performed by v 2 test using SPSS software (SPSS Inc, Chicago, Ill). Survival rate was calculated by the Kaplan-Meier method, and statistical significance was examined using the log-rank test.
Fig. 2 A, Synaptophysin staining. Chromogranin was stained in the cytoplasm of a few tumor cells (200). Pancreatic islet was also strongly stained (arrow). B, Tumor cells were diffusely and strongly stained to chromogranin in the cytoplasm of tumor cells (400).
Neuroendocrine differentiation
735
3.2. Immunohistochemical staining in EBD carcinomas Expression of synaptophysin and chromogranin to tumor cells was seen in the cytoplasm of tumor cells. Synaptophysin and chromogranin were also stained in the cytoplasm of pancreatic islet cells and nerve fibers in the outer part of the smooth muscle or fibrous tissue of the bile duct, or of the pancreas (Fig. 2). There was no staining of synaptophysin or chromogranin in the adjacent normal bile duct epithelial cells. Synaptophysin and chromogranin were positive in 54 (27.8%) and 74 (38.1%) cases, respectively. Thirtyfour cases (17.5%) were positive for both synaptophysin and chromogranin. Twenty (10.3%) and 40 cases (20.6%) were positive only for synaptophysin and for chromogranin, respectively. One hundred cases (51.6%) were negative for both markers. Of the 194 examined cases, 94 cases showed neuroendocrine differentiation (48.4%) with either synaptophysin or chromogranin positivity or positivity in both.
1.0
Combined expression Syn(+)/Chr(+) Syn(+)/Chr(-) Syn(-)/Chr(+) Syn(-)/Chr(-)
.8
Cumulative survival
subtypes showed 155 cases of adenocarcinoma, not otherwise specified, 18 cases of papillary carcinomas, 9 cases of intestinal-type adenocarcinomas, 5 cases of adenosquamous carcinomas, and 4 cases of mucinous carcinomas. There was 1 case each of clear cell, signet ring cell, and sarcomatoid carcinoma. Cases with neuroendocrine features, such as carcinoid tumors and small cell carcinomas, were not observed.
.6
.4
.2
0.0 0
24
48
72
96
120
144
Survival (months) Fig. 4 Kaplan-Meier survival analysis based on synaptophysin and/or chromogranin immunostaining status. There was significant survival difference between Syn (+)/Chr (+) (median survival, 21 months) versus Syn ( )/Chr (+) (median survival, 38 months) (log-rank test, P = .035) and Syn (+)/Chr (+) versus Syn ( )/Chr ( ) (median survival, 35 months) ( P = .035). However, there was no survival difference between Syn (+)/Chr ( ) (median survival, 28 months) versus Syn ( )/Chr (+), Syn (+)/Chr (+) versus Syn (+)/Chr ( ), Syn (+)/Chr ( ) versus Syn ( )/Chr ( ), and Syn ( )/Chr (+) versus Syn ( )/Chr ( ). Chr indicates chromogranin.
3.3. Correlation between neuroendocrine differentiation and clinicopathologic variables
1.0 Syn expression Presence Absence
Cumulative survival
.8
.6
.4
.2
0.0 0
24
48
72
96
120
144
Survival (months) Fig. 3 Kaplan-Meier survival analysis based on synaptophysin positivity (log-rank test, P = .015). Median survival of synaptophysin positive group was 27 months, whereas that of synaptophysin negative group was 38 months. Syn indicates synaptophysin.
Clinicopathologic variables were compared with synaptophysin positivity, chromogranin positivity, combined synaptophysin and chromogranin positivity, and numbers of neuroendocrine marker expression. There were no significant differences among synaptophysin- or chromogranin-only positivity, combined synaptophysin and chromogranin positivity, or numbers of neuroendocrine marker expression and clinicopathologic variables such as age, sex, tumor location, size, growth pattern and microscopic subtypes, depth of invasion, metastasis to lymph node, vascular and perineural invasion, or hepatic, pancreatic, and duodenal invasion. Chromogranin positivity was more frequently seen in intestinal-type adenocarcinomas (77.8%, 7/9 cases) than in adenocarcinomas, not otherwise specified (34.2%, 53/155 cases), and in papillary carcinomas (33.3%, 6/18 cases; P = .029). Although v 2 test was not performed in several carcinomas because of the small number of cases, 3 of 4 mucinous carcinomas, 3 of 5 adenosquamous carcinomas, and each case of clear cell and signet ring cell carcinoma were positive to chromogranin. The presence of overall neuroendocrine differentiation was more common in female (60.0%, 33/55 cases) than in
736
S.-M. Hong et al. No. of NE marker (+) 2 markers expression 1 marker expression
Cumulative survival
.8
No marker expression
.6
.4
.2
0.0 0
24
48
72
96
120
144
Survival (months) Fig. 5 Kaplan-Meier survival analysis based on number of neuroendocrine marker expression. Median survival of the 2–neuroendocrine marker expression group was 21 months, whereas that of the no–neuroendocrine marker expression group was 35 months (log-rank test, P = .035). However, there was no survival difference between the 2–neuroendocrine marker expression and 1–neuroendocrine marker expression groups (median survival, 38 months) ( P = .062). NE indicates neuroendocrine.
male patients (43.8%, 61/139 cases; P = .031). There was no significant difference in the presence or absence of neuroendocrine differentiation related to other clinicopathologic variables. No significant difference was found between the degree of synaptophysin positivity and clinicopathologic variables. However, there was a significant difference between the degree of chromogranin positivity and depth of invasion ( P = .021) and pancreatic invasion ( P = .017). EBD carcinomas with pancreatic invasion showed a higher degree of chromogranin expression than those without pancreatic invasion. EBD carcinomas with deeper invasion had more positive tumor cells for chromogranin than those with superficial invasion. There was no significant difference between the degree of chromogranin positivity and other clinicopathologic variables.
3.4. Neuroendocrine differentiation and patients’ survival
Regarding overall neuroendocrine differentiation, which was defined by either synaptophysin or chromogranin positivity, or both, there was no significant survival difference between the presence and absence of neuroendocrine differentiation. A significant survival difference was observed between the group with both synaptophysin and chromogranin expression and the group with no expression to both neuroendocrine markers. The median survival time was significantly shorter in carcinomas that were positive for both synaptophysin and chromogranin (21 months) than in carcinomas that were negative for both synaptophysin and chromogranin (35 months; P = .035) or in tumors that were positive for chromogranin but negative for synaptophysin (38 months; P = .035; Fig. 4). However, no survival difference was found between synaptophysin-only expression group (28 months) and both synaptophysin and chromogranin expression group (21 months). When the numbers of neuroendocrine marker expression and survival were compared, there was a significant survival difference between the 2–neuroendocrine marker expression and the no–neuroendocrine marker expression groups ( P = .035; Fig. 5). The median survival of cases with the 2–neuroendocrine marker expression group was 21 months, whereas that of the no–neuroendocrine marker expression group was 35 months. There was no statistical difference between the 2–neuroendocrine marker expression group and the 1–neuroendocrine marker expression group or between the 1–neuroendocrine marker expression group and the no–neuroendocrine marker expression group. There was no significant survival difference between the degree of synaptophysin positivity and the survival rate. 1.0
Chr degree Degree 2 Degree 1
.8
Cumulative survival
1.0
.6
.4
.2
0.0 0
There was a significant survival difference between overall synaptophysin-positive (median survival, 27 months) and synaptophysin-negative (median survival, 38 months) groups ( P = .015; Fig. 3). However, no statistically significant difference was found between overall chromogranin-positive and chromogranin-negative groups.
24
48
72
96
120
144
Survival (months) Fig. 6 Kaplan-Meier survival analysis based on degree of chromogranin positivity. Median survival of degree 1 was 38 months, whereas that of degree 2 was 21 months (log-rank test, P = .016).
Neuroendocrine differentiation
737
Table 1 Univariate analysis of the prognostic factors in 194 cases of EBD carcinomas Variable (no. of cases, total N = 194) Age V60 (n = 87) N60 (n = 107) Sex Male (n = 139) Female (n = 55) Growth pattern Papillary (n = 25) Nodular (n = 17) Infiltrative (n = 152) Size b2 cm (n = 83) 2-4 cm (n = 93) N4 cm (n = 18) T classification T1 (n = 40) T2 (n = 65) T3 (n = 79) T4 (n = 10) Duodenal invasion Absent (n = 184) Present (n = 10) Hepatic invasion Absent (n = 184) Present (n = 10) Pancreatic invasion Absent (n = 116) Present (n = 78) Perineural invasion Absent (n = 65) Present (n = 129) Vascular invasion Absent (n = 147) Present (n = 47) Marginal involvement Absent (n = 150) Present (n = 44) Lymph node metastasis Absent (n = 132) Present (n = 62)
Median survival (mo)
P
39 29
.026T
34 27
.370
53 –a 28
.013T
43 27 38
.341
–a 29 27 13
b.001T
35 13
.003T
33 18
.231
41 27
.025T
44 29
.087
41 18
b.001T
38 20
.023T
44 18
b.001T
a Median survival could not be obtained because more than 50% of the cases survived after a 5-year follow-up. T Significant at the b.05 level.
However, a significant survival difference was found between the degree of chromogranin positivity and the survival rate ( P = .016; Fig. 6). The median survival of degrees 1 and 2 of chromogranin positivity was 38 and 30 months, respectively. A univariate analysis showed that the age of the patients (60 and b60 years old versus N60 years old; P = .026), growth type ( P = .013), T classification ( P b .001), involvement of resection margin ( P = .023), metastasis to lymph node ( P b .001), and pancreatic
( P = .025), duodenal ( P = .003), and vascular invasion ( P b .001), as well as synaptophysin positivity, degree of chromogranin expression, dual expression of synaptophysin and chromogranin, and numbers of neuroendocrine marker expression, were significantly related to overall survival (Table 1). The prognostic significance of synaptophysin positivity, degree of chromogranin positivity and expression, dual expression of synaptophysin and chromogranin, and number of neuroendocrine marker expression were further analyzed by the Cox proportional hazard model along with other significant clinicopathologic variables. A multivariate analysis revealed that synaptophysin expression showed a strong independent prognostic significance ( P = .017; Table 2) along with the age of the patients ( P = .029), T classification ( P = .010), vascular invasion ( P = .018), and metastasis to lymph nodes ( P = .004). However, the degree of chromogranin expression, the dual expression of synaptophysin and chromogranin, and the numbers of neuroendocrine marker expression were not independent prognostic factors. We additionally investigated the effect of the neuroendocrine expression for T classification and for the status of lymph node metastasis to evaluate whether the effect of the neuroendocrine differentiation is confounded with that of the T and N classifications, because T and N classifications appeared to be important prognostic factors in the present study (Table 3). When lymph node metastasis was absent (N0 classification), there was a significant survival difference between synaptophysinpositive (median, 29 months) and synaptophysin-negative (60 months) groups ( P b .001). When tumor invaded the pancreas or liver (T3 classification), there was a significant survival difference between chromogranin-positive
Table 2 Multivariate analysis of the prognostic factors in 194 cases of EBD carcinomas Variable
P
Relative risk
95% CI
Age T classification Lymph node metastasis Vascular invasion Synaptophysin expression Degree of chromogranin expression Combined expression of synaptophysin and chromogranin Number of neuroendocrine marker
.029T .010T .004T
1.537 2.294 1.801
1.045-2.260 1.308-4.024 1.212-2.677
.018T .017T
1.618 1.572
1.085-2.415 1.082-2.278
.523
0.370
0.127-1.080
.280
1.357
0.782-2.354
.430
1.209
0.692-2.114
T Significant at the b.05 level.
738
S.-M. Hong et al.
Table 3 Survival analysis according to the neuroendocrine differentiation after adjusting for T and N classifications in 194 cases of EBD carcinomas Variable (total N = 194)
T classification T1 (n = 40) T2 (n = 65) T3 (n = 79) T4 (n = 10) N classification N0 (n = 132) N1 (n = 62)
Median survival (mo) (no. of cases) Synaptophysin nonexpression
Synaptophysin expression
–a (n = 32) 30 (n = 46) 29 (n = 56) 13 (n = 6)
–a 23 22 13
60 (n = 92) 18 (n = 48)
29 (n = 40) 18 (n = 14)
(n (n (n (n
= = = =
8) 19) 23) 4)
P
.843 .162 .149 .605 b.001T .674
Median survival (mo) (no. of cases)
P
Chromogranin nonexpression
Chromogranin expression
51 29 35 30
23) 41) 49) 7)
–a (n = 17) 29 (n = 24) 19 (n = 30) 11 (n = 3)
.274 .937 .032T .210
44 (n = 85) 19 (n = 35)
39 (n = 47) 18 (n = 27)
.589 .231
(n (n (n (n
= = = =
a
Median survival could not be obtained because more than 50% of the cases survived after a 5-year follow-up. T Significant at the b.05 level.
(median, 19 months) and chromogranin-negative (35 months) groups ( P = .032).
4. Discussion EBD carcinoma is an uncommon neoplasm. Although the tumor is usually detected early with jaundice because of obstruction of the bile duct by the tumor before distant metastasis, the survival rate is dismal. The median survival time of 2 to 5 years of localized carcinomas has been reported to be up to 40% [1]. In addition to already established prognostic factors [1-8], including histological type and grade, site of tumor, depth of invasion, duodenal and pancreatic invasion, involvement of the resection margin, and vascular, lymphatic, and perineural invasion, new factors affecting patients’ survival, such as K-ras mutation [26], have been reported. Neuroendocrine differentiation, which is characterized by single or scattered clusters of neuroendocrine cells in morphologically nonneuroendocrine neoplasms [27], is reported to be a prognostic factor in certain carcinomas [9-17]. Whereas neuroendocrine neoplasms, including carcinoid and small cell carcinoma, show neuroendocrine features including organoid nests, palisading and trabecular patterns, as well as rosettelike structures on hematoxylineosin–stained slides [28]. Tumors with neuroendocrine differentiation and neuroendocrine neoplasms are usually determined by light microscopic features and by immunoreactivity for certain neuroendocrine markers including neuron-specific enolase (NSE), synaptophysin, chromogranin, CD56, or ectopic bioactive hormones such as serotonin and somatostatin. In the present study, we analyzed neuroendocrine differentiation in EBD carcinomas and its impact on the patients’ survival. In this study, synaptophysin and chromogranin were used for defining neuroendocrine differentiation because they have been recommended to be the most reliable markers by the World Health Organization [28]. We did not use NSE immunostaining
because it is known to be nonspecific for neuroendocrine markers [29]. The frequency of neuroendocrine differentiation of colonic, gastric, and pancreatic adenocarcinomas has been reported to be 4% to 40%, 10% to 40%, and 8% to 45%, respectively [16,18,30,31]. However, the neuroendocrine differentiation in EBD carcinomas was not well explored. The frequency of neuroendocrine differentiation in EBD carcinomas in 2 previous studies reported completely different results [24,25]. A Japanese study [25] reported that 22.7% of EBD carcinomas revealed neuroendocrine differentiation, whereas in a Western study [24], it was 76.9% (10/13 cases). In the present study, 48.5% (94/194 cases) of EBD carcinomas expressed neuroendocrine differentiation, which was determined by synaptophysin or chromogranin positivity, or both synaptophysin- and chromogranin-positive immunostaining. Synaptophysin and chromogranin positivity were 27.8% (54/194 cases) and 38.1% (74/194 cases), respectively. These results were quite different from the study of Hsu et al [24], which reported 61.5% positivity in synaptophysin expression (8/13 cases) and 0% (0/13 cases) positivity in chromogranin expression in EBD carcinomas. Yamamoto et al [25] used gastrin and somatostatin in their study, whereas Hsu et al [24] used NSE, chromogranin A, synaptophysin, serotonin, substance P, somatostatin, and glucagons in their study. The difference in neuroendocrine positivity results may be because of the use of different antibodies and/or to technical advances, such as antigen retrieval technique. The prognostic impact of neuroendocrine differentiation in EBD carcinomas has been rarely analyzed. A previous study [24] classified tumors of the gallbladder and the EBD on the basis of immunohistochemical results as pure exocrine carcinoma, predominantly exocrine carcinoma with occasional neuroendocrine cells, mixed exocrine and endocrine carcinoma, predominantly neuroendocrine carcinoma with occasional exocrine cells, and pure neuroendocrine carcinoma. They then analyzed the prognostic significance according to the degree of neuroendocrine
Neuroendocrine differentiation differentiation and reported that the survival time among patients with pure neuroendocrine or predominantly neuroendocrine carcinomas (2.6 months) was significantly shorter than that of predominantly exocrine carcinoma with occasional neuroendocrine cells or mixed exocrineneuroendocrine carcinomas (13.5 months). Because of the small number of cases, a statistical analysis for patients’ survival was not performed [24]; therefore, their results should be interpreted as preliminary information rather than a solid conclusion. In the present study with 194 cases of EBD carcinomas, we demonstrated that synaptophysin expression, a higher degree of chromogranin expression, and dual expression to synaptophysin and chromogranin impacted poorly on patients’ survival. Although overall chromogranin expression did not affect patients’ survival, it affected poorly on patients’ survival in T3 tumors. When lymph node metastasis was absent (N0 classification), synaptophysin expression also showed worse patients’ outcome. Like neuroendocrine cells of the gastrointestinal tract, those in bile ducts have been reported to secrete a variety of peptide hormones that have significant growth factor activity [32,33]. The poorer prognosis of EBD carcinomas with neuroendocrine differentiation, especially in those with expression to synaptophysin, may reflect either autocrine or paracrine stimulation, or both, by neurosecretory products of tumor cells. A possible explanation is that in EBD carcinomas, synaptophysin may contribute more than chromogranin to autocrine or paracrine stimulation. In cases with synaptophysin expression, an additional assessment of chromogranin expression may potentiate the predictability of patients’ survival (21 months of median survival in both expressions; 28 months in synaptophysin-only positive). The reason for poorer prognosis of carcinomas with dual expression to synaptophysin and chromogranin was unclear. It is likely that the 2–neuroendocrine marker expression group may have contained a higher component of cells with autocrine and/or paracrine function than the 1–neuroendocrine marker expression group, and therefore, more neurosecretory products could be secreted. However, more studies are required to document the significance of neuroendocrine differentiation in the survival rate of patients with EBD carcinoma. The present study showed a significant survival difference among different degrees of chromogranin positivity. The higher the proportion of chromogranin positive tumor cells in EBD carcinomas, the poorer the survival rate. These results were similar to those of the previous study [24] and supported our postulation of a poorer survival rate in the 2–neuroendocrine marker expression group by more autocrine and/or paracrine stimulation. We performed univariate and multivariate analyses to examine the effect of neuroendocrine differentiation on the survival of patients with EBD carcinomas. Univariate and multivariate analyses revealed that synaptophysin expression was an important prognostic predictor for patients with
739 EBD carcinomas, especially when lymph node metastasis was absent. In a comparison of chromogranin expression with histological subtypes, intestinal-type adenocarcinoma showed more frequent chromogranin positivity than adenocarcinoma, nitric oxide synthase, and papillary carcinoma. As described in the previous study [25], neuroendocrine differentiation was predominantly observed in well-differentiated to moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas in the present study. The cases with a higher degree of chromogranin-positive tumor cells had a tendency to invade deeply and showed more frequent pancreatic invasion. Neuroendocrine differentiation was observed more frequently in female than in male patients. The cause of more frequent neuroendocrine differentiation in females was unclear, and additional studies, including hormonal effect, are required to clarify the mechanism and the significance. In EBD carcinomas in which the tumor has invaded the pancreas, the assessment of neuroendocrine differentiation should be meticulous, because the islet cells in the pancreas can be mistakenly interpreted as tumor cells with synaptophysin and chromogranin positivity (Fig. 2A). When we had cases with tumor cells invading the pancreas, matched hematoxylineosin–stained slides were meticulously examined to not include entrapped islet cells as neuroendocrine-positive tumor cells. In addition, neuroendocrine-positive tumor cells were morphologically different from islet cells; the islet cells showed regular, round nuclei with finely granular chromatin, whereas tumor cells demonstrated irregular, pleomorphic nuclei with chromatin clumping and clearing, and mitotic features. In summary, neuroendocrine differentiation in EBD carcinomas was 48.5%. Chromogranin-positive carcinomas (38.1%) were more common than synaptophysin-positive carcinomas (27.8%). Although there was no statistically significant difference between clinicopathologic parameters and synaptophysin positivity, synaptophysin immunohistochemical results revealed an important prognostic factor. Synaptophysin-positive cases showed a worse prognosis than synaptophysin-negative cases. Although chromogranin status alone failed to show prognostic significance, tumors with more chromogranin-positive cells and with dual expression to both synaptophysin and chromogranin exhibited a worse prognosis than those with synaptophysin expression alone. Therefore, additional immunohistochemical studies with synaptophysin and chromogranin may be useful for the evaluation of the biological behavior of EBD carcinomas.
Acknowledgments This work was presented, in part, at the 93rd Annual Meeting of the United States and Canadian Academy
740 of Pathology, Vancouver, British Columbia, Canada, March 2004.
References [1] Greene FL, Page DL, Fleming ID, et al. AJCC cancer staging manual. 6th ed. New York7 Springer; 2002. [2] Bhuiya MR, Nimura Y, Kamiya J, Kondo S, Nagino M, Hayakawa N. Clinicopathologic features influencing survival of patients with bile duct carcinoma. Multivariate statistical analysis. World J Surg 1993; 17:653 - 7. [3] Chung C, Bautista N, O’Connell TX. Prognosis and treatment of bile duct carcinoma. Am Surg 1998;64:921 - 5. [4] Davis RI, Sloan JM, Hood JM, Maxwell P. Carcinoma of the extrahepatic biliary tract: clinicopathological and immunohistochemical study. Histopathology 1992;12:623 - 31. [5] Henson DE, Albores-Saavedra J, Corle D. Carcinoma of the extrahepatic bile ducts: histologic types, stage of disease, grade, and survival rates. Cancer 1992;70:1491 - 501. [6] Longmire WP, McArthur MS, Bastounis EA, Hiatt J. Carcinoma of the extrahepatic biliary tract. Ann Surg 1973;178:333 - 45. [7] Ouchi K, Suzuki M, Hashimoto L, Sato T. Histologic findings and prognostic factors in carcinoma of the upper bile duct. Am J Surg 1989;157:552 - 6. [8] Tompkins RK, Thomas D, Wile A, Longmire Jr WP. Prognostic factors in the bile duct carcinoma: analysis of 96 cases. Ann Surg 1981;194:447 - 55. [9] Grabowski P, Schindler I, Anagnostopoulos I, et al. Neuroendocrine differentiation is a relevant prognostic factor in stage III-IV colorectal cancer. Eur J Gastroenterol Hepatol 2001;3:405 - 11. [10] Weinstein MH, Partin AW, Veltri RW, Epstein JI. Neuroendocrine differentiation in prostate cancer: enhanced prediction of progression after radical prostatectomy. Hum Pathol 1996;27:683 - 7. [11] Bollito E, Berruti A, Bellina M, et al. Relationship between neuroendocrine features and prognostic parameters in human prostate adenocarcinoma. Ann Oncol 2001;12(Suppl 2):S159 - 64. [12] Rasmuson T, Grankvist K, Roos G, Ljungberg B. Neuroendocrine differentiation in renal cell carcinoma. Acta Oncologica 1999;38: 623 - 8. [13] Tseleni-Balafouta S, Kavantzas N, Alevizaki M, Paraskevakou H, Davaris P. Neuroendocrine differentiation in follicular-thyroid carcinoma: correlation to prognosis factors in papillary carcinoma. J Exp Clin Cancer Res 1998;17:533 - 7. [14] Carles J, Rosell R, Ariza A, et al. Neuroendocrine differentiation as a prognostic factor in non–small cell lung cancer. Lung Cancer 1993; 10:209 - 19. [15] Schleusener JT, Tazelaar HD, Jung SH, et al. Neuroendocrine differentiation is an independent prognostic factor in chemotherapytreated nonsmall cell lung carcinoma. Cancer 1996;77:1284 - 91.
S.-M. Hong et al. [16] Saclarides TJ, Szeluga D, Staren ED. Neuroendocrine cancers of the colon and rectum. Results of a ten-year experience. Dis Colon Rectum 1994;37:635 - 42. [17] Indinnimeo M, Cicchini C, Memeo L, et al. Correlation between chromogranin-A expression and pathological variables in human colon carcinoma. Anticancer Res 2002;22:395 - 8. [18] Tezel E, Nagasaka T, Nomoto S, Sugimoto H, Nakao A. Neuroendocrine-like differentiation in patients with pancreatic carcinoma. Cancer 2000;89:2230 - 6. [19] Miremadi A, Pinder SE, Lee AH, et al. Neuroendocrine differentiation and prognosis in breast adenocarcinoma. Histopathology 2002;40: 215 - 22. [20] Blumenfeld W, Chandhoke DK, Sagerman P, Turi GK. Neuroendocrine differentiation in gastric adenocarcinoma: an immunohistochemical study. Arch Pathol Lab Med 1996;120:478 - 81. [21] Melissari M, Giordano G, Saragoni L, Bordi C. Comparative study of endocrine differentiation in elderly and advanced stomach carcinoma. Pathologica 1997;89:133 - 7. [22] Hamilton K, Chiappori A, Olson S, Sawyers J, Johnson D, Washington K. Prevalence and prognostic significance of neuroendocrine cells in esophageal adenocarcinoma. Mod Pathol 2000;13: 475 - 81. [23] Savargaonkar PR, Hale RJ, Mutton A, Manning V, Buckley CH. Neuroendocrine differentiation in cervical carcinoma. J Clin Pathol 1996;49:139 - 41. [24] Hsu W, Deziel DJ, Gould VE, Warren WH, Gooch GT, Staren ED. Neuroendocrine differentiation and prognosis of extrahepatic biliary tract carcinoma. Surgery 1991;110:604 - 10. [25] Yamamoto M, Takahashi I, Iwamoto T, Mandai K, Tahara E. Endocrine cells in the extrahepatic bile duct carcinoma. J Cancer Res Clin Oncol 1984;108:331 - 5. [26] Malats N, Porta M, Pinol J, Corominas JM, Real FX. Ki-ras mutations as a prognostic factor in extrahepatic bile system cancer. PANK-ras I Project Investigators. J Clin Oncol 1995;13:1679 - 86. [27] Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma. Prostate 1999;39:135 - 48. [28] Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological typing of lung and pleural tumors. 3rd ed. Berlin7 Springer; 1999. [29] Dranoff G, Bigner DD. A word of caution in the use of neuronspecific enolase in tumor diagnosis. Arch Pathol Lab Med 1984; 108:535. [30] Yao GY, Zhou JL, Lai MD, Chen XQ, Chen PH. Neuroendocrine markers in adenocarcinomas: an investigation of 356 cases. World J Gastroenterol 2003;9:858 - 61. [31] Waldum HL, Aase S, Kvetnoi I, et al. Neuroendocrine differentiation in human gastric carcinoma. Cancer 1998;83:435 - 44. [32] Johns LR. Physiology of the gastrointestinal tract. 2nd ed. New York7 Raven; 1987. [33] Zachary I, Woll PJ, Rozengurt E. A role for neuropeptides in the control of cell proliferation. Dev Biol 1987;124:295 - 308.
www.elsevier.com/locate/humpath
Neuroendocrine differentiation in extrahepatic bile duct carcinomas and its prognostic significance Seung-Mo Hong MD, PhD, Mi-Jung Kim MD, David Y. Pi, Daniel Jo, Eunsil Yu MD, PhD, Jae Y. Ro MD, PhD*,1 Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, South Korea Received 21 December 2004; accepted 3 May 2005
Keywords: Extrahepatic bile duct carcinoma; Immunohistochemistry; Neuroendocrine differentiation; Synaptophysin; Chromogranin
Summary Neuroendocrine differentiation is known to be one of the prognostic factors in many carcinomas. However, the characteristics of neuroendocrine differentiation are not well elucidated in extrahepatic bile duct (EBD) carcinomas. One hundred ninety-four cases of EBD carcinomas were analyzed using immunohistochemistry with synaptophysin and chromogranin. The tumors were graded as degree 0, 1, and 2 when the positive tumor cells were 5% or less, 6% to 25%, and 26% or more, respectively. Immunohistochemical results were compared with clinicopathologic variables and survival rate. Synaptophysin and chromogranin were positive in 54 (27.8%) and 74 (38.1%) cases, respectively. Thirty-four cases (17.5%) were positive for both synaptophysin and chromogranin, 20 (10.3%) and 40 cases (20.6%) were positive only for synaptophysin and for chromogranin, respectively, and 100 cases (51.6%) were negative for both markers. There was a significant survival difference between overall synaptophysin-positive (median, 27 months) and synaptophysin-negative (38 months) groups ( P b .05). However, there was no survival difference between chromograninpositive and chromogranin-negative groups. There was a significant survival difference between the dual-positive expression to synaptophysin and chromogranin group (median, 21 months) and the dualnegative expression group (median, 35 months; P b .05). In summary, synaptophysin expression was an important prognostic factor because synaptophysin-positive cases showed a worse prognosis than synaptophysin-negative cases. The more tumor cells expressed chromogranin, the poorer the survival. Therefore, immunohistochemical studies for neuroendocrine differentiation may be helpful in routine pathological examinations for evaluating the survival and the prognosis of patients with EBD carcinomas. D 2005 Elsevier Inc. All rights reserved.
1. Introduction
T Corresponding author. E-mail addresses: [email protected], [email protected] (J.Y. Ro). 1 Current address: Department of Pathology, The Methodist Hospital, 6565 Fannin Street, Houston, TX 77030. 0046-8177/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2005.05.002
Extrahepatic bile duct (EBD) carcinoma is an uncommon neoplasm with a dismal prognosis. Two- to 5-year median survival of localized EBD carcinoma was reported to be about 20% to 40% [1]. Several factors have been demonstrated as prognostic factors, such as histological type,
Neuroendocrine differentiation histological grade, site of tumor, depth of invasion, duodenal and pancreatic invasion, involvement of the resection margin, and vascular, lymphatic, and perineural invasion [1-8]. Neuroendocrine differentiation has been reported in neoplasms from many different organs with a variable prognostic significance. It is reported to be one of the worst prognostic factors in prostate [9-11], kidney [12], thyroid gland [13], lung [14,15], and colon [16,17] carcinomas. On the other hand, the presence of neuroendocrine differentiation showed a better survival in pancreatic carcinoma [18], and no prognostic significance in other neoplasms such as breast [19], stomach [20,21], esophagus [22], and cervix [23] carcinomas. Although a few small series have been reported, neuroendocrine differentiation in EBD carcinomas is not well characterized [24,25], and clinicopathologic significance of the neuroendocrine differentiation is still uncertain. A previous study from a Western country reported that neuroendocrine differentiation was associated with a shorter survival rate [24]; however, this study is not fully accepted because of the limited number of cases. Therefore, an additional clinicopathologic study on neuroendocrine differentiation in EBD carcinomas with a large number of cases is required to validate the issue. In this study, we analyzed neuroendocrine differentiation in a large number of EBD carcinomas and compared the results with clinicopathologic parameters to clarify whether neuroendocrine differentiation has a prognostic impact to the patients.
2. Materials and methods 2.1. Case selection One hundred ninety-four surgically resected cases with available tissue blocks for immunostaining of EBD carcinoma from the files of the Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea, from 1991 to 2000, were included in this study. Only carcinomas with the epicenter in the EBD were included in this study. Either carcinomas with the epicenter in the ampulla of Vater or pancreas or carcinomas with obvious precancerous epithelial changes in the ampulla of Vater or pancreas were excluded. In addition, equivocal cases in terms of origin of the tumors were also excluded from this study. Carcinomas arising from the gallbladder and intrahepatic bile duct with extension to the EBD were also excluded from this study. Data obtained from reviewing medical records included patient’s age, sex, survival time, and survival status, and from pathology reports were tumor location, size, and growth pattern. Information on postoperative radiation and/or chemotherapy and performance status of patients were not analyzed in this study.
2.2. Pathological study Microscopic investigation was made on the hematoxylineosin–stained slides. Histological type, depth of tumor
733 invasion, metastasis to lymph nodes, vascular and perineural invasion, involvement of the resection margin, and involvement of the pancreas, duodenum, and liver were evaluated.
2.3. Immunohistochemical staining A representative paraffin-embedded tissue block from each case was selected for immunohistochemical study. Immunohistochemical staining was carried out by the streptoavidin-biotin method, using a commercially available kit. Deparaffinized sections were treated with methanol containing 3% H2O2 for 10 minutes after 2 times antigen retrieval using a microwave processor at 958C for 5 minutes. After washing with phosphate-buffered saline, blocking serum was applied for 10 minutes. Then, primary antibodies, synaptophysin (antimouse synaptophysin monoclonal antibody, Dinnona, Inc, Seoul, South Korea; 1:200 dilution), and chromogranin (antimouse chromogranin monoclonal antibody, Dinnona; 1:200 dilution) were allowed to react at room temperature for 1 hour. After washing in phosphate-buffered saline, biotin-marked secondary antibody was applied for 10 minutes followed by peroxidase-marked streptoavidin for 10 minutes on both antibodies. The reaction was visualized by diaminobenzidine tetrahydrochloride. Nuclei were stained with Harris hematoxylin. Sections from normal pancreas containing islet cells were used as positive controls for synaptophysin and chromogranin. Negative controls were performed by substituting a nonimmune purified mouse monoclonal antibody for the primary antibodies.
2.4. Interpretation of immunohistochemical staining The immunoreactivity for each case was interpreted as positive and negative, and then graded as degree 0, 1, and 2 when 5% or less, 6% to 25%, and 26% or more, respectively, of the tumor cells were positive for synaptophysin and chromogranin (Fig. 1). We counted positive cells among 500 cells in the most active areas and provided actual numbers of positive percentage.
2.5. Comparison of neuroendocrine differentiation with clinicopathologic parameters Immunohistochemical results of the synaptophysin and chromogranin were compared with clinicopathologic variables, which included age, sex, location, size, growth patterns, histological types, T classification, marginal status, nodal metastasis, survival rate, and vascular, perineural, pancreatic, hepatic, and duodenal invasion. The presence of neuroendocrine differentiation was defined as any or both neuroendocrine markers positivity and was compared with the abovementioned clinicopathologic variables. Expression to both synaptophysin and chromogranin, to synaptophysin only, and to chromogranin only and no expression to either synaptophysin or chromogranin
734
S.-M. Hong et al. Prognostic factors were tested by univariate and multivariate analyses (proportional hazards regression model). P b .05 was considered statistically significant.
3. Results 3.1. Clinicopathologic characteristics of cases The ages of the patients ranged from 30 to 84 years (mean, 60 years; SD, 10 years). One hundred thirty-nine cases were men and 55 were women. The tumors showed infiltrative growth pattern in 152 cases, papillary in 25, and nodular in 17 cases. The tumor size was less than 2 cm in 83 cases, between 2 and 4 cm in 93 cases, and more than 4 cm in 18 cases. There were 98 proximal (perihilar) tumors, 88 distal, and 8 diffuse tumors. Forty cases were confined within the bile duct (T1 classification), 65 cases invaded the fibroadipose tissue (T2 classification), 79 were T3 tumors, and 10 were T4 tumors. In addition, there were 10 cases of hepatic invasions, 78 pancreatic invasions, 129 perineural invasions, and 47 vascular invasions. Tumor cells involved the resection margins in 44 cases, whereas 62 cases showed metastasis to lymph nodes. Histological
Fig. 1 Degree of the immunohistochemical staining. A, No tumor cells were stained with synaptophysin (degree 0, 200). B, Ten percent of tumor cells were positive for synaptophysin (degree 1, 200). C, Seventy percent of tumor cells were positive for synaptophysin (degree 2, 200).
were also compared with the abovementioned clinicopathologic variables. After that, coexpression versus no expression to both markers was compared with the clinicopathologic variables.
2.6. Statistical analyses Statistical analyses were performed by v 2 test using SPSS software (SPSS Inc, Chicago, Ill). Survival rate was calculated by the Kaplan-Meier method, and statistical significance was examined using the log-rank test.
Fig. 2 A, Synaptophysin staining. Chromogranin was stained in the cytoplasm of a few tumor cells (200). Pancreatic islet was also strongly stained (arrow). B, Tumor cells were diffusely and strongly stained to chromogranin in the cytoplasm of tumor cells (400).
Neuroendocrine differentiation
735
3.2. Immunohistochemical staining in EBD carcinomas Expression of synaptophysin and chromogranin to tumor cells was seen in the cytoplasm of tumor cells. Synaptophysin and chromogranin were also stained in the cytoplasm of pancreatic islet cells and nerve fibers in the outer part of the smooth muscle or fibrous tissue of the bile duct, or of the pancreas (Fig. 2). There was no staining of synaptophysin or chromogranin in the adjacent normal bile duct epithelial cells. Synaptophysin and chromogranin were positive in 54 (27.8%) and 74 (38.1%) cases, respectively. Thirtyfour cases (17.5%) were positive for both synaptophysin and chromogranin. Twenty (10.3%) and 40 cases (20.6%) were positive only for synaptophysin and for chromogranin, respectively. One hundred cases (51.6%) were negative for both markers. Of the 194 examined cases, 94 cases showed neuroendocrine differentiation (48.4%) with either synaptophysin or chromogranin positivity or positivity in both.
1.0
Combined expression Syn(+)/Chr(+) Syn(+)/Chr(-) Syn(-)/Chr(+) Syn(-)/Chr(-)
.8
Cumulative survival
subtypes showed 155 cases of adenocarcinoma, not otherwise specified, 18 cases of papillary carcinomas, 9 cases of intestinal-type adenocarcinomas, 5 cases of adenosquamous carcinomas, and 4 cases of mucinous carcinomas. There was 1 case each of clear cell, signet ring cell, and sarcomatoid carcinoma. Cases with neuroendocrine features, such as carcinoid tumors and small cell carcinomas, were not observed.
.6
.4
.2
0.0 0
24
48
72
96
120
144
Survival (months) Fig. 4 Kaplan-Meier survival analysis based on synaptophysin and/or chromogranin immunostaining status. There was significant survival difference between Syn (+)/Chr (+) (median survival, 21 months) versus Syn ( )/Chr (+) (median survival, 38 months) (log-rank test, P = .035) and Syn (+)/Chr (+) versus Syn ( )/Chr ( ) (median survival, 35 months) ( P = .035). However, there was no survival difference between Syn (+)/Chr ( ) (median survival, 28 months) versus Syn ( )/Chr (+), Syn (+)/Chr (+) versus Syn (+)/Chr ( ), Syn (+)/Chr ( ) versus Syn ( )/Chr ( ), and Syn ( )/Chr (+) versus Syn ( )/Chr ( ). Chr indicates chromogranin.
3.3. Correlation between neuroendocrine differentiation and clinicopathologic variables
1.0 Syn expression Presence Absence
Cumulative survival
.8
.6
.4
.2
0.0 0
24
48
72
96
120
144
Survival (months) Fig. 3 Kaplan-Meier survival analysis based on synaptophysin positivity (log-rank test, P = .015). Median survival of synaptophysin positive group was 27 months, whereas that of synaptophysin negative group was 38 months. Syn indicates synaptophysin.
Clinicopathologic variables were compared with synaptophysin positivity, chromogranin positivity, combined synaptophysin and chromogranin positivity, and numbers of neuroendocrine marker expression. There were no significant differences among synaptophysin- or chromogranin-only positivity, combined synaptophysin and chromogranin positivity, or numbers of neuroendocrine marker expression and clinicopathologic variables such as age, sex, tumor location, size, growth pattern and microscopic subtypes, depth of invasion, metastasis to lymph node, vascular and perineural invasion, or hepatic, pancreatic, and duodenal invasion. Chromogranin positivity was more frequently seen in intestinal-type adenocarcinomas (77.8%, 7/9 cases) than in adenocarcinomas, not otherwise specified (34.2%, 53/155 cases), and in papillary carcinomas (33.3%, 6/18 cases; P = .029). Although v 2 test was not performed in several carcinomas because of the small number of cases, 3 of 4 mucinous carcinomas, 3 of 5 adenosquamous carcinomas, and each case of clear cell and signet ring cell carcinoma were positive to chromogranin. The presence of overall neuroendocrine differentiation was more common in female (60.0%, 33/55 cases) than in
736
S.-M. Hong et al. No. of NE marker (+) 2 markers expression 1 marker expression
Cumulative survival
.8
No marker expression
.6
.4
.2
0.0 0
24
48
72
96
120
144
Survival (months) Fig. 5 Kaplan-Meier survival analysis based on number of neuroendocrine marker expression. Median survival of the 2–neuroendocrine marker expression group was 21 months, whereas that of the no–neuroendocrine marker expression group was 35 months (log-rank test, P = .035). However, there was no survival difference between the 2–neuroendocrine marker expression and 1–neuroendocrine marker expression groups (median survival, 38 months) ( P = .062). NE indicates neuroendocrine.
male patients (43.8%, 61/139 cases; P = .031). There was no significant difference in the presence or absence of neuroendocrine differentiation related to other clinicopathologic variables. No significant difference was found between the degree of synaptophysin positivity and clinicopathologic variables. However, there was a significant difference between the degree of chromogranin positivity and depth of invasion ( P = .021) and pancreatic invasion ( P = .017). EBD carcinomas with pancreatic invasion showed a higher degree of chromogranin expression than those without pancreatic invasion. EBD carcinomas with deeper invasion had more positive tumor cells for chromogranin than those with superficial invasion. There was no significant difference between the degree of chromogranin positivity and other clinicopathologic variables.
3.4. Neuroendocrine differentiation and patients’ survival
Regarding overall neuroendocrine differentiation, which was defined by either synaptophysin or chromogranin positivity, or both, there was no significant survival difference between the presence and absence of neuroendocrine differentiation. A significant survival difference was observed between the group with both synaptophysin and chromogranin expression and the group with no expression to both neuroendocrine markers. The median survival time was significantly shorter in carcinomas that were positive for both synaptophysin and chromogranin (21 months) than in carcinomas that were negative for both synaptophysin and chromogranin (35 months; P = .035) or in tumors that were positive for chromogranin but negative for synaptophysin (38 months; P = .035; Fig. 4). However, no survival difference was found between synaptophysin-only expression group (28 months) and both synaptophysin and chromogranin expression group (21 months). When the numbers of neuroendocrine marker expression and survival were compared, there was a significant survival difference between the 2–neuroendocrine marker expression and the no–neuroendocrine marker expression groups ( P = .035; Fig. 5). The median survival of cases with the 2–neuroendocrine marker expression group was 21 months, whereas that of the no–neuroendocrine marker expression group was 35 months. There was no statistical difference between the 2–neuroendocrine marker expression group and the 1–neuroendocrine marker expression group or between the 1–neuroendocrine marker expression group and the no–neuroendocrine marker expression group. There was no significant survival difference between the degree of synaptophysin positivity and the survival rate. 1.0
Chr degree Degree 2 Degree 1
.8
Cumulative survival
1.0
.6
.4
.2
0.0 0
There was a significant survival difference between overall synaptophysin-positive (median survival, 27 months) and synaptophysin-negative (median survival, 38 months) groups ( P = .015; Fig. 3). However, no statistically significant difference was found between overall chromogranin-positive and chromogranin-negative groups.
24
48
72
96
120
144
Survival (months) Fig. 6 Kaplan-Meier survival analysis based on degree of chromogranin positivity. Median survival of degree 1 was 38 months, whereas that of degree 2 was 21 months (log-rank test, P = .016).
Neuroendocrine differentiation
737
Table 1 Univariate analysis of the prognostic factors in 194 cases of EBD carcinomas Variable (no. of cases, total N = 194) Age V60 (n = 87) N60 (n = 107) Sex Male (n = 139) Female (n = 55) Growth pattern Papillary (n = 25) Nodular (n = 17) Infiltrative (n = 152) Size b2 cm (n = 83) 2-4 cm (n = 93) N4 cm (n = 18) T classification T1 (n = 40) T2 (n = 65) T3 (n = 79) T4 (n = 10) Duodenal invasion Absent (n = 184) Present (n = 10) Hepatic invasion Absent (n = 184) Present (n = 10) Pancreatic invasion Absent (n = 116) Present (n = 78) Perineural invasion Absent (n = 65) Present (n = 129) Vascular invasion Absent (n = 147) Present (n = 47) Marginal involvement Absent (n = 150) Present (n = 44) Lymph node metastasis Absent (n = 132) Present (n = 62)
Median survival (mo)
P
39 29
.026T
34 27
.370
53 –a 28
.013T
43 27 38
.341
–a 29 27 13
b.001T
35 13
.003T
33 18
.231
41 27
.025T
44 29
.087
41 18
b.001T
38 20
.023T
44 18
b.001T
a Median survival could not be obtained because more than 50% of the cases survived after a 5-year follow-up. T Significant at the b.05 level.
However, a significant survival difference was found between the degree of chromogranin positivity and the survival rate ( P = .016; Fig. 6). The median survival of degrees 1 and 2 of chromogranin positivity was 38 and 30 months, respectively. A univariate analysis showed that the age of the patients (60 and b60 years old versus N60 years old; P = .026), growth type ( P = .013), T classification ( P b .001), involvement of resection margin ( P = .023), metastasis to lymph node ( P b .001), and pancreatic
( P = .025), duodenal ( P = .003), and vascular invasion ( P b .001), as well as synaptophysin positivity, degree of chromogranin expression, dual expression of synaptophysin and chromogranin, and numbers of neuroendocrine marker expression, were significantly related to overall survival (Table 1). The prognostic significance of synaptophysin positivity, degree of chromogranin positivity and expression, dual expression of synaptophysin and chromogranin, and number of neuroendocrine marker expression were further analyzed by the Cox proportional hazard model along with other significant clinicopathologic variables. A multivariate analysis revealed that synaptophysin expression showed a strong independent prognostic significance ( P = .017; Table 2) along with the age of the patients ( P = .029), T classification ( P = .010), vascular invasion ( P = .018), and metastasis to lymph nodes ( P = .004). However, the degree of chromogranin expression, the dual expression of synaptophysin and chromogranin, and the numbers of neuroendocrine marker expression were not independent prognostic factors. We additionally investigated the effect of the neuroendocrine expression for T classification and for the status of lymph node metastasis to evaluate whether the effect of the neuroendocrine differentiation is confounded with that of the T and N classifications, because T and N classifications appeared to be important prognostic factors in the present study (Table 3). When lymph node metastasis was absent (N0 classification), there was a significant survival difference between synaptophysinpositive (median, 29 months) and synaptophysin-negative (60 months) groups ( P b .001). When tumor invaded the pancreas or liver (T3 classification), there was a significant survival difference between chromogranin-positive
Table 2 Multivariate analysis of the prognostic factors in 194 cases of EBD carcinomas Variable
P
Relative risk
95% CI
Age T classification Lymph node metastasis Vascular invasion Synaptophysin expression Degree of chromogranin expression Combined expression of synaptophysin and chromogranin Number of neuroendocrine marker
.029T .010T .004T
1.537 2.294 1.801
1.045-2.260 1.308-4.024 1.212-2.677
.018T .017T
1.618 1.572
1.085-2.415 1.082-2.278
.523
0.370
0.127-1.080
.280
1.357
0.782-2.354
.430
1.209
0.692-2.114
T Significant at the b.05 level.
738
S.-M. Hong et al.
Table 3 Survival analysis according to the neuroendocrine differentiation after adjusting for T and N classifications in 194 cases of EBD carcinomas Variable (total N = 194)
T classification T1 (n = 40) T2 (n = 65) T3 (n = 79) T4 (n = 10) N classification N0 (n = 132) N1 (n = 62)
Median survival (mo) (no. of cases) Synaptophysin nonexpression
Synaptophysin expression
–a (n = 32) 30 (n = 46) 29 (n = 56) 13 (n = 6)
–a 23 22 13
60 (n = 92) 18 (n = 48)
29 (n = 40) 18 (n = 14)
(n (n (n (n
= = = =
8) 19) 23) 4)
P
.843 .162 .149 .605 b.001T .674
Median survival (mo) (no. of cases)
P
Chromogranin nonexpression
Chromogranin expression
51 29 35 30
23) 41) 49) 7)
–a (n = 17) 29 (n = 24) 19 (n = 30) 11 (n = 3)
.274 .937 .032T .210
44 (n = 85) 19 (n = 35)
39 (n = 47) 18 (n = 27)
.589 .231
(n (n (n (n
= = = =
a
Median survival could not be obtained because more than 50% of the cases survived after a 5-year follow-up. T Significant at the b.05 level.
(median, 19 months) and chromogranin-negative (35 months) groups ( P = .032).
4. Discussion EBD carcinoma is an uncommon neoplasm. Although the tumor is usually detected early with jaundice because of obstruction of the bile duct by the tumor before distant metastasis, the survival rate is dismal. The median survival time of 2 to 5 years of localized carcinomas has been reported to be up to 40% [1]. In addition to already established prognostic factors [1-8], including histological type and grade, site of tumor, depth of invasion, duodenal and pancreatic invasion, involvement of the resection margin, and vascular, lymphatic, and perineural invasion, new factors affecting patients’ survival, such as K-ras mutation [26], have been reported. Neuroendocrine differentiation, which is characterized by single or scattered clusters of neuroendocrine cells in morphologically nonneuroendocrine neoplasms [27], is reported to be a prognostic factor in certain carcinomas [9-17]. Whereas neuroendocrine neoplasms, including carcinoid and small cell carcinoma, show neuroendocrine features including organoid nests, palisading and trabecular patterns, as well as rosettelike structures on hematoxylineosin–stained slides [28]. Tumors with neuroendocrine differentiation and neuroendocrine neoplasms are usually determined by light microscopic features and by immunoreactivity for certain neuroendocrine markers including neuron-specific enolase (NSE), synaptophysin, chromogranin, CD56, or ectopic bioactive hormones such as serotonin and somatostatin. In the present study, we analyzed neuroendocrine differentiation in EBD carcinomas and its impact on the patients’ survival. In this study, synaptophysin and chromogranin were used for defining neuroendocrine differentiation because they have been recommended to be the most reliable markers by the World Health Organization [28]. We did not use NSE immunostaining
because it is known to be nonspecific for neuroendocrine markers [29]. The frequency of neuroendocrine differentiation of colonic, gastric, and pancreatic adenocarcinomas has been reported to be 4% to 40%, 10% to 40%, and 8% to 45%, respectively [16,18,30,31]. However, the neuroendocrine differentiation in EBD carcinomas was not well explored. The frequency of neuroendocrine differentiation in EBD carcinomas in 2 previous studies reported completely different results [24,25]. A Japanese study [25] reported that 22.7% of EBD carcinomas revealed neuroendocrine differentiation, whereas in a Western study [24], it was 76.9% (10/13 cases). In the present study, 48.5% (94/194 cases) of EBD carcinomas expressed neuroendocrine differentiation, which was determined by synaptophysin or chromogranin positivity, or both synaptophysin- and chromogranin-positive immunostaining. Synaptophysin and chromogranin positivity were 27.8% (54/194 cases) and 38.1% (74/194 cases), respectively. These results were quite different from the study of Hsu et al [24], which reported 61.5% positivity in synaptophysin expression (8/13 cases) and 0% (0/13 cases) positivity in chromogranin expression in EBD carcinomas. Yamamoto et al [25] used gastrin and somatostatin in their study, whereas Hsu et al [24] used NSE, chromogranin A, synaptophysin, serotonin, substance P, somatostatin, and glucagons in their study. The difference in neuroendocrine positivity results may be because of the use of different antibodies and/or to technical advances, such as antigen retrieval technique. The prognostic impact of neuroendocrine differentiation in EBD carcinomas has been rarely analyzed. A previous study [24] classified tumors of the gallbladder and the EBD on the basis of immunohistochemical results as pure exocrine carcinoma, predominantly exocrine carcinoma with occasional neuroendocrine cells, mixed exocrine and endocrine carcinoma, predominantly neuroendocrine carcinoma with occasional exocrine cells, and pure neuroendocrine carcinoma. They then analyzed the prognostic significance according to the degree of neuroendocrine
Neuroendocrine differentiation differentiation and reported that the survival time among patients with pure neuroendocrine or predominantly neuroendocrine carcinomas (2.6 months) was significantly shorter than that of predominantly exocrine carcinoma with occasional neuroendocrine cells or mixed exocrineneuroendocrine carcinomas (13.5 months). Because of the small number of cases, a statistical analysis for patients’ survival was not performed [24]; therefore, their results should be interpreted as preliminary information rather than a solid conclusion. In the present study with 194 cases of EBD carcinomas, we demonstrated that synaptophysin expression, a higher degree of chromogranin expression, and dual expression to synaptophysin and chromogranin impacted poorly on patients’ survival. Although overall chromogranin expression did not affect patients’ survival, it affected poorly on patients’ survival in T3 tumors. When lymph node metastasis was absent (N0 classification), synaptophysin expression also showed worse patients’ outcome. Like neuroendocrine cells of the gastrointestinal tract, those in bile ducts have been reported to secrete a variety of peptide hormones that have significant growth factor activity [32,33]. The poorer prognosis of EBD carcinomas with neuroendocrine differentiation, especially in those with expression to synaptophysin, may reflect either autocrine or paracrine stimulation, or both, by neurosecretory products of tumor cells. A possible explanation is that in EBD carcinomas, synaptophysin may contribute more than chromogranin to autocrine or paracrine stimulation. In cases with synaptophysin expression, an additional assessment of chromogranin expression may potentiate the predictability of patients’ survival (21 months of median survival in both expressions; 28 months in synaptophysin-only positive). The reason for poorer prognosis of carcinomas with dual expression to synaptophysin and chromogranin was unclear. It is likely that the 2–neuroendocrine marker expression group may have contained a higher component of cells with autocrine and/or paracrine function than the 1–neuroendocrine marker expression group, and therefore, more neurosecretory products could be secreted. However, more studies are required to document the significance of neuroendocrine differentiation in the survival rate of patients with EBD carcinoma. The present study showed a significant survival difference among different degrees of chromogranin positivity. The higher the proportion of chromogranin positive tumor cells in EBD carcinomas, the poorer the survival rate. These results were similar to those of the previous study [24] and supported our postulation of a poorer survival rate in the 2–neuroendocrine marker expression group by more autocrine and/or paracrine stimulation. We performed univariate and multivariate analyses to examine the effect of neuroendocrine differentiation on the survival of patients with EBD carcinomas. Univariate and multivariate analyses revealed that synaptophysin expression was an important prognostic predictor for patients with
739 EBD carcinomas, especially when lymph node metastasis was absent. In a comparison of chromogranin expression with histological subtypes, intestinal-type adenocarcinoma showed more frequent chromogranin positivity than adenocarcinoma, nitric oxide synthase, and papillary carcinoma. As described in the previous study [25], neuroendocrine differentiation was predominantly observed in well-differentiated to moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas in the present study. The cases with a higher degree of chromogranin-positive tumor cells had a tendency to invade deeply and showed more frequent pancreatic invasion. Neuroendocrine differentiation was observed more frequently in female than in male patients. The cause of more frequent neuroendocrine differentiation in females was unclear, and additional studies, including hormonal effect, are required to clarify the mechanism and the significance. In EBD carcinomas in which the tumor has invaded the pancreas, the assessment of neuroendocrine differentiation should be meticulous, because the islet cells in the pancreas can be mistakenly interpreted as tumor cells with synaptophysin and chromogranin positivity (Fig. 2A). When we had cases with tumor cells invading the pancreas, matched hematoxylineosin–stained slides were meticulously examined to not include entrapped islet cells as neuroendocrine-positive tumor cells. In addition, neuroendocrine-positive tumor cells were morphologically different from islet cells; the islet cells showed regular, round nuclei with finely granular chromatin, whereas tumor cells demonstrated irregular, pleomorphic nuclei with chromatin clumping and clearing, and mitotic features. In summary, neuroendocrine differentiation in EBD carcinomas was 48.5%. Chromogranin-positive carcinomas (38.1%) were more common than synaptophysin-positive carcinomas (27.8%). Although there was no statistically significant difference between clinicopathologic parameters and synaptophysin positivity, synaptophysin immunohistochemical results revealed an important prognostic factor. Synaptophysin-positive cases showed a worse prognosis than synaptophysin-negative cases. Although chromogranin status alone failed to show prognostic significance, tumors with more chromogranin-positive cells and with dual expression to both synaptophysin and chromogranin exhibited a worse prognosis than those with synaptophysin expression alone. Therefore, additional immunohistochemical studies with synaptophysin and chromogranin may be useful for the evaluation of the biological behavior of EBD carcinomas.
Acknowledgments This work was presented, in part, at the 93rd Annual Meeting of the United States and Canadian Academy
740 of Pathology, Vancouver, British Columbia, Canada, March 2004.
References [1] Greene FL, Page DL, Fleming ID, et al. AJCC cancer staging manual. 6th ed. New York7 Springer; 2002. [2] Bhuiya MR, Nimura Y, Kamiya J, Kondo S, Nagino M, Hayakawa N. Clinicopathologic features influencing survival of patients with bile duct carcinoma. Multivariate statistical analysis. World J Surg 1993; 17:653 - 7. [3] Chung C, Bautista N, O’Connell TX. Prognosis and treatment of bile duct carcinoma. Am Surg 1998;64:921 - 5. [4] Davis RI, Sloan JM, Hood JM, Maxwell P. Carcinoma of the extrahepatic biliary tract: clinicopathological and immunohistochemical study. Histopathology 1992;12:623 - 31. [5] Henson DE, Albores-Saavedra J, Corle D. Carcinoma of the extrahepatic bile ducts: histologic types, stage of disease, grade, and survival rates. Cancer 1992;70:1491 - 501. [6] Longmire WP, McArthur MS, Bastounis EA, Hiatt J. Carcinoma of the extrahepatic biliary tract. Ann Surg 1973;178:333 - 45. [7] Ouchi K, Suzuki M, Hashimoto L, Sato T. Histologic findings and prognostic factors in carcinoma of the upper bile duct. Am J Surg 1989;157:552 - 6. [8] Tompkins RK, Thomas D, Wile A, Longmire Jr WP. Prognostic factors in the bile duct carcinoma: analysis of 96 cases. Ann Surg 1981;194:447 - 55. [9] Grabowski P, Schindler I, Anagnostopoulos I, et al. Neuroendocrine differentiation is a relevant prognostic factor in stage III-IV colorectal cancer. Eur J Gastroenterol Hepatol 2001;3:405 - 11. [10] Weinstein MH, Partin AW, Veltri RW, Epstein JI. Neuroendocrine differentiation in prostate cancer: enhanced prediction of progression after radical prostatectomy. Hum Pathol 1996;27:683 - 7. [11] Bollito E, Berruti A, Bellina M, et al. Relationship between neuroendocrine features and prognostic parameters in human prostate adenocarcinoma. Ann Oncol 2001;12(Suppl 2):S159 - 64. [12] Rasmuson T, Grankvist K, Roos G, Ljungberg B. Neuroendocrine differentiation in renal cell carcinoma. Acta Oncologica 1999;38: 623 - 8. [13] Tseleni-Balafouta S, Kavantzas N, Alevizaki M, Paraskevakou H, Davaris P. Neuroendocrine differentiation in follicular-thyroid carcinoma: correlation to prognosis factors in papillary carcinoma. J Exp Clin Cancer Res 1998;17:533 - 7. [14] Carles J, Rosell R, Ariza A, et al. Neuroendocrine differentiation as a prognostic factor in non–small cell lung cancer. Lung Cancer 1993; 10:209 - 19. [15] Schleusener JT, Tazelaar HD, Jung SH, et al. Neuroendocrine differentiation is an independent prognostic factor in chemotherapytreated nonsmall cell lung carcinoma. Cancer 1996;77:1284 - 91.
S.-M. Hong et al. [16] Saclarides TJ, Szeluga D, Staren ED. Neuroendocrine cancers of the colon and rectum. Results of a ten-year experience. Dis Colon Rectum 1994;37:635 - 42. [17] Indinnimeo M, Cicchini C, Memeo L, et al. Correlation between chromogranin-A expression and pathological variables in human colon carcinoma. Anticancer Res 2002;22:395 - 8. [18] Tezel E, Nagasaka T, Nomoto S, Sugimoto H, Nakao A. Neuroendocrine-like differentiation in patients with pancreatic carcinoma. Cancer 2000;89:2230 - 6. [19] Miremadi A, Pinder SE, Lee AH, et al. Neuroendocrine differentiation and prognosis in breast adenocarcinoma. Histopathology 2002;40: 215 - 22. [20] Blumenfeld W, Chandhoke DK, Sagerman P, Turi GK. Neuroendocrine differentiation in gastric adenocarcinoma: an immunohistochemical study. Arch Pathol Lab Med 1996;120:478 - 81. [21] Melissari M, Giordano G, Saragoni L, Bordi C. Comparative study of endocrine differentiation in elderly and advanced stomach carcinoma. Pathologica 1997;89:133 - 7. [22] Hamilton K, Chiappori A, Olson S, Sawyers J, Johnson D, Washington K. Prevalence and prognostic significance of neuroendocrine cells in esophageal adenocarcinoma. Mod Pathol 2000;13: 475 - 81. [23] Savargaonkar PR, Hale RJ, Mutton A, Manning V, Buckley CH. Neuroendocrine differentiation in cervical carcinoma. J Clin Pathol 1996;49:139 - 41. [24] Hsu W, Deziel DJ, Gould VE, Warren WH, Gooch GT, Staren ED. Neuroendocrine differentiation and prognosis of extrahepatic biliary tract carcinoma. Surgery 1991;110:604 - 10. [25] Yamamoto M, Takahashi I, Iwamoto T, Mandai K, Tahara E. Endocrine cells in the extrahepatic bile duct carcinoma. J Cancer Res Clin Oncol 1984;108:331 - 5. [26] Malats N, Porta M, Pinol J, Corominas JM, Real FX. Ki-ras mutations as a prognostic factor in extrahepatic bile system cancer. PANK-ras I Project Investigators. J Clin Oncol 1995;13:1679 - 86. [27] Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma. Prostate 1999;39:135 - 48. [28] Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological typing of lung and pleural tumors. 3rd ed. Berlin7 Springer; 1999. [29] Dranoff G, Bigner DD. A word of caution in the use of neuronspecific enolase in tumor diagnosis. Arch Pathol Lab Med 1984; 108:535. [30] Yao GY, Zhou JL, Lai MD, Chen XQ, Chen PH. Neuroendocrine markers in adenocarcinomas: an investigation of 356 cases. World J Gastroenterol 2003;9:858 - 61. [31] Waldum HL, Aase S, Kvetnoi I, et al. Neuroendocrine differentiation in human gastric carcinoma. Cancer 1998;83:435 - 44. [32] Johns LR. Physiology of the gastrointestinal tract. 2nd ed. New York7 Raven; 1987. [33] Zachary I, Woll PJ, Rozengurt E. A role for neuropeptides in the control of cell proliferation. Dev Biol 1987;124:295 - 308.