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Immunohistochemical prognostic indicators of gastrointestinal carcinoid tumours
EJSO 2002; 28: 140–146 doi:10.1053/ejso.2001.1229, available online at http://www.idealibrary.com on
Immunohistochemical prognostic indicators of gastrointestinal carcinoid tumours M. Kawahara∗, M. Kammori∗, H. Kanauchi∗, C. Noguchi∗, S. Kuramoto∗, M. Kaminishi∗, H. Endo† and K. Takubo‡ Departments of ∗ Gastroenterological Surgery and † Pathology, University of Tokyo and ‡ Department of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
Aims: The aim of this study was to determine whether expression of the oncoproteins p21, p53, E-cadherin (EC), cyclin D1, bcl-2 and Rb and the proliferation marker Ki-67 is predictive of malignant behaviour in gastrointestinal carcinoid tumours. Methods: Immunohistochemical (IHC) staining was performed on carcinoid tumours from 41 patients (31 rectal, eight gastrointestinal, two appendiceal lesions). The six tumours that had invaded deeply into the muscularis propria or beyond, had metastasized to regional lymph nodes or had metastasized to a distant site were classified as the malignant group, and the other 35 tumours formed the benign group. IHC expression was compared between the two groups, and the prognostic value of each marker was assessed. Results: Of the six tumours in the malignant group, 66.7% were p21 positive, 0% were p53 positive, 33.3% were EC positive, 100% were cyclin D1 positive, 33.3% were Rb positive, 16.7% were bcl-2 positive and 50% were Ki-67 positive. Of the 35 tumours in the benign group, 17.1% were p21 positive, 0% were p53 positive, 100% were EC positive, 94.3% were cyclin D1 positive, 8.6% were Rb positive, 17.1% were bcl-2 positive and 0% were Ki-67 positive. Conclusions: These data show that p53, cyclin D1, Rb, bcl-2 and Ki-67 staining does not correlate with malignant behaviour but that overexpression of p21 (P=0.02) and reduced staining of EC (P=0.005) do correlate with malignant behaviour. These two parameters may therefore be useful as prognostic indicators for gastrointestinal carcinoid tumours. 2002 Elsevier Science Ltd Key words: carcinoid tumour; immunohistochemistry; oncoprotein.
INTRODUCTION Carcinoid tumours are uncommon neoplasms that arise from neuroendocrine cells and occur most commonly in the gastrointestinal tract. They may produce a wide variety of biologically active agents. Although they may metastasize, their behaviour is very variable,1–3 and conventional classifications, including tumour, node and metastasis staging, are rather unsatisfactory because of the variation in survival within each entity. At present, little is known about the biological parameters that predict the disease course of carcinoid tumours.4–7 Treatment options for gastrointestinal carcinoid tumours include enterectomy with lymph node resection,8 local surgical excision9 and endoscopic resection.10,11 As
Correspondence to: Masaki Kawahara, MD, Department of Gastroenterological Surgery, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. Tel: +81-3-3815-5411; Fax: +813-5800-9734; E-mail: [email protected] 0748–7983/02/020140+07 $35.00/0
noted above, however, the behaviour of these tumours is variable, and criteria for selecting the most appropriate therapeutic approach have yet to be established. Identification of prognostic indicators that can be determined histologically might provide guidance for selecting optimal treatment of these tumours. The prognosis and choice of therapy for human neoplasms are determined by the stage of the disease and their likely behaviour. At present, their likely behaviour is estimated by routine histopathological examination of tissue sections obtained from the primary lesion. However, traditional histopathological findings are insufficient to identify patients with gastrointestinal neoplasms who are at risk of metastasis or recurrence.12 Molecular phenotyping has recently provided a useful extra dimension for characterization of biological potential, and it can be of assistance in predicting of clinical outcome. In this study, we assessed several oncoproteins that correlate with tumorigenesis in order to evaluate the malignant potential of gastrointestinal carcinoid tumours. 2002 Elsevier Science Ltd
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Table 1 Characteristics of the 41 patients with gastrointestinal carcinoids Characteristics Sex Location of tumour
Depth of invasion Lymph node metastasis Distant metastasis Vessel invasion Treatment
Number of patients Male Female Rectum Appendix Stomach, duodenum -sm pm+ − + − + − Surgical resection Endoscopic resection
27 14 31 2 8 38 3 3 38 1 40 4 37 38 3
-sm, mucosa or submucosa; pm-, tumour invasion to the muscularis propria or deeper layer; +, positive; −, negative; vessel invasion, lymphatic and/or venous invasion.
The objectives of the study were (a) to characterize the expression of p21, p53, E-cadherin (EC), cyclin D1, bcl-2, Rb, and Ki-67 in gastrointestinal carcinoid tumours by immunohistochemistry (IHC), (b) to investigate the significance of these oncoproteins by comparing IHC results in ‘malignant’ and ‘benign’ lesions and (c) to assess the prognostic value of expression of these markers.
MATERIALS AND METHODS Tissues and patient characteristics Archival tissues from 41 patients with gastrointestinal carcinoid tumours who had undergone surgical (n=38) or endoscopic (n=3) resection in our department or an affiliated hospital between January 1982 and December 1998 were used. Briefly, the patients’ mean age was 60 years (range: 39–86 years), and the male:female ratio was 27:14. Thirty-one patients had a rectal carcinoid, eight had a gastroduodenal carcinoid, and two had an appendiceal carcinoid (Table 1). In this series ‘malignant’ carcinoid tumours was defined as a carcinoid tumour with deep invasion into the muscularis propria or beyond, regional lymph node metastasis or distant haematogenous spread. Carcinoid tumours limited to the submucosal layer, without regional lymphatic or distant metastasis, were classified as benign.
IHC staining Sections of paraffin-embedded blocks from the carcinoid tumours were studied by IHC using monoclonal antibodies to p21 (1:20 dilution, Clone EA10; Oncogene Research Products, Cambridge, MA, USA), p53 (1:100 dilution, Clone DO-7; DACO, Glostrup, Denmark), EC
(1:50 dilution, Clone HECD-1, Takara Shuzo, Shiga, Japan), cyclin D1 (1:10 dilution, Clone DSC-6; PROGEN, Heidelberg, Germany), Rb (1:50 dilution, Clone 3H9; MBL, Nagoya, Japan), bcl-2 (1:50 dilution, Clone 124; DACO, Glostrup, Denmark) and Ki-67 (1:50 dilution, Clone Mib-1; IMMUNOTECH, Marseille, France), with the avidin–biotin complex (ABC) method. Briefly, 5 m thick sections were cut from the blocks, deparaffinized with xylene and dehydrated through graded concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. The sections were then treated with microwave radiation for 10 min for antigen retrieval, and, to block intrinsic antibody binding, they were reacted with normal serum (mouse IgG) for 10 min at room temperature. After the tissue sections had been exposed to primary antibodies overnight at 4°C, with appropriate negative and positive controls, they were reacted with biotinylated anti-mouse antibody (secondary antibody) for 10 min and with ABC for another 10 min, with intervening washes. Diaminobenzidine tetrahydrochloride was used as the final chromogen, and sections were counterstained with Mayer’s haematoxylin before mounting.
Specimen analysis IHC evaluation was conducted by two independent investigators (M.K. and K.T.), who scored the estimated percentage of tumour cells that exhibited nuclear, cytoplasmic and cell membrane staining. The percentage of immunoreactive tumour cells for each primary antibody used was evaluated. Positivity for each antibody was defined as reported previously.13–16 Tumour cells were considered strongly positive (2+) for p53 (nuclear
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Table 2 Category definitions for positive immunoreactivity Stain
p53 p21 bcl-2 EC CycD1 Ki-67 Rb
Positive
Negative
Strong
Intermediate
>50% >50% >50% >90% overexpression [10% [10% Overexpression
5–50% 5–50% 5–50% 10–90%
<5% <5% <5% <5% <10% <10% No expression
CycD1, cyclin D1; over- and no expression were defined by comparing with the internal control.
staining), p21 (nuclear staining), or bcl-2 (cytoplasmic staining) if >50% showed strong reactivity, intermediately positive (1+) if between 5% and 50% were immunoreactive and negative (−) if <5% were immunoreactive. EC (cytoplasmic and membrane staining) was regarded as strongly positive (2+) if >90% of tumour cells showed stronger expression than the surrounding non-neoplastic tissue, intermediately positive (1+) if 10–90% were immunoreactive and negative (−) if <10% were immunoreactive. Tumour cells were considered positive for cyclin D1 (nuclear staining) and Ki-67 (nuclear staining) when [10% of the cells were immunoreactive. Staining for Rb was scored as Rb negative if none of the tumour cells exhibited nuclear staining and the Rb-positive cells in background tissue showed adequate nuclear staining as a positive internal control (Table 2). The IHC analysis was performed blind, without knowledge of the clinical data.
Statistical analysis Logistic regression was used for univariate and multivariate categorical analysis of IHC staining. P<0.05 was considered significant.
RESULTS Pathological and clinical characteristics Patient characteristics and the histopathological findings in the 41 tumours are listed in Table 1. Of the 41 tumours, three showed deep invasion, three had metastasized to lymph nodes and one had metastasized to the liver. A total of six tumours with metastasis or deep invasion were classified in the malignant group (Table 3).
IHC staining for p53, cyclin D1, Rb and bcl-2 No tumours showed positive staining for p53 protein. Cyclin D1 (2+) protein was detected in most of the
tumours, 100% of the malignant group and 94.3% of the benign group, but staining was generally stronger in the former (Fig. 1). Rb expression was variable: 33.3% (two of six) were positive in the malignant group and 8.6% (three of 35) were positive in the benign group. Bcl-2 was little different between the two groups: 16.7% (one of six) were intermediately positive (1+) in the malignant group and 17.1% (six of 35) intermediate positive (1+) in the benign group. No tumours showed strong positive staining for bcl-2 protein. Accordingly, there was no significant correlation between p53, cyclin D1, Rb or bcl-2 expression and the malignant behaviour of the carcinoid tumours (Table 4).
IHC staining of p21, EC and Ki-67 Immunoreactivity, indicative of p21 protein overexpression (1+) (Fig. 2), was found in four of the six tumours (66.7%) in the malignant group, vs six of the 35 tumours (17.1%) in the control group. The difference was significant (P<0.05) with 5% positive expression as the cut-off point.14,16 Two of the six tumours (33.3%) in the malignant group showed strongly positive (2+) cytoplasmic and membrane staining for EC (Fig. 3) and four (66.7%) were negative (−), whereas 100% of the tumours in the benign group showed strong positive (2+) expression (Fig. 4). We therefore employed 90% positive expression as the cut-off point for EC. This difference was also statistically significant (P<0.05), and the carcinoid cells in the benign group showed much stronger expression than those in the malignant group. Tumour cell nuclei were considered positive for Ki67 antigen when they showed dark brown granular staining. More than threshold staining was evident in 50.0% (three of six) of the malignant group but in none of the tumours in the benign group. The difference, however, did not reach statistial significance. One case of gastroduodenal carcinoid with liver metastases (case 4 in Table 3) had a high Ki-67-positive rate (Fig. 5), and
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Table 3 Summary of details for the six patients with malignant carcinoids Number
Age (years)
Sex
Location
Size (mm)
Depth
Lymph node metastasis
Distant metastasis
Outcome
46 61 69 51 86 77
F M M M M M
R R R D T A
15 9 8 22 62 60
pm sm sm pm ss pm
− + + + − +
− − − + − −
Alive Alive Alive ED NED NED
1 2 3 4 5 6
F, female; M, male; R, rectum; D, duodenum; T, transverse colon; A, appendix; pm, muscularis propria; sm, submucosa; ss, subserosa; +, positive; −, negative; ED, dead with evidence of disease; NED, dead with no evidence of disease.
Figure 1 Cyclin D1 immunostain of a carcinoid tumour of the rectum demonstrating strong immunoreactivity of tumour in comparison with the adjacent non-neoplastic glandular cells (×100).
Figure 2 p21 immunostain showing positive immunoreactivity of tumour cells of a duodenal carcinoid tumour with a malignant phenotype (×200).
Table 4 Summary of data for immunostaining of the carcinoid tumours Stain
p53 p21 EC CycD1 Ki-67 bcl-2 Rb
Malignant group (n=6)
Benign group (n=35)
Positive
Negative
Positive
Negative
P value
0% 66.7% 33.3% 100% 50.0% 16.7% 33.3%
100% 33.3% 66.7% 0% 50.0% 83.7% 66.7%
0% 17.1% 100% 94.3% 0% 17.1% 8.6%
100% 82.9% 0% 5.7% 100% 82.9% 91.4%
1.00 0.02 0.005 0.94 0.09 0.88 0.15
CycD1, cyclin D1.
the patient was treated by distal gastrectomy and partial hepatectomy, but died of recurrent liver metastases. There was no interaction between parameters when the categorical data for IHC expression were subjected to multivariate logistic analysis.
DISCUSSION Carcinoid tumours are generally characterized by slower evolution and lower grade malignant behaviour than carcinomas. However, it is well known that carcinoid
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Figure 3 EC immunostain showing negative reactivity of tumour cells of a rectal carcinoid tumour with a malignant phenotype (×100).
Figure 4 EC immunostain showing positive reactivity of tumour cells of a rectal carcinoid tumour with a benign phenotype (×100).
Figure 5 Ki-67 immunostain demonstrating strong nuclear positivity of tumour cells of a gastric carcinoid tumour (×200).
tumours may metastasize, and optimal treatment is controversial because of the difficulty in identifying features of aggressive behaviour. A number of parameters
M. KAWAHARA ET AL. have been proposed for predicting the clinical behaviour and outcome of carcinoid tumours, and tumour size, depth of invasion and histological appearance have been considered the most reliable.8–10 Nevertheless, the behaviour of this group of tumours still remains unpredictable, and this sometimes leads to difficulty in selecting the most appropriate treatment for an individual patient. More reliable prognostic indicators for carcinoid tumours are therefore required. Recent advances in molecular biology and in our comprehension of carcinogenesis and tumour development have provided new tools with which to determine the metastatic potential of human malignancies. The p53 gene encodes a 53 kDa nuclear phosphoprotein that is involved in the regulation of cell proliferation and apoptosis. Mutation of the p53 gene results in neoplastic transformation in a wide variety of human organs and tissues, as typified by carcinoma of the colon.13–18 However, we found no significant correlation between p53 overexpression and malignant behaviour in gastrointestinal carcinoid tumours in the present study. This is in agreement with the findings in a previous report on colorectal carcinoid tumours.19 The oncoprotein for p21 is a p53 downstream protein and plays a key role in the p53 suppressor function that controls the G1–S cell cycle checkpoint. Since the p21 gene is transcriptionally activated by wild-type p53, alteration of p53 function may result in loss of p21 expression, and this may be one of the mechanisms by which altered p53 influences tumour progression.14,16–18,20 Conversely, overexpression of p21 is considered to lead to inhibition of cellular growth and proliferation.14,17,20 Although our study showed no p53 overexpression, IHC staining of p21 protein, an indicator of overexpression, was significantly more common in the malignant group than in the benign group. This was unexpected in view of the negative p53 staining, but it has been demonstrated that p21 expression can also be mediated through p53-independent pathways.14,21,22 EC, a member of the cadherin family of cell surface glycoproteins, is a calcium-dependent cell-to-cell adhesion molecule and invasion suppressor molecule that is found in epithelial tissues. It effects its adhesive function via the homotopic interactions of five extracellular repeated domains, with its intracellular portion complexing with several distinct undercoat proteins, including members of the catenin family. Loss of EC function is closely related to malignant behaviour, which is characterized by dedifferentiation, invasion, metastasis and overproliferation.12,13,22–26 We observed overexpression of EC in all the benign carcinoid tumours in this series, and it may be possible to use EC overexpression as an IHC marker for the diagnosis of carcinoid tumours. However, this needs to be studied in many more cases. We also observed a significant correlation between markedly reduced expression of
IMMUNOHISTOCHEMISTRY OF GASTROINTESTINAL CARCINOID TUMOURS EC and degree of malignancy, suggesting a relationship between loss of cell adhesion and progression of carcinoid tumours. In other words, EC overexpression appears to be a favourable prognostic indicator for carcinoid tumours. To our knowledge this is the first report of an association between EC expression, as determined by IHC, and the prognosis of carcinoid tumours. Cyclin D1 is a 45 kDa cell cycle regulatory protein that is transcribed from the CCND1 gene at 11q13 and acts at the late G1 checkpoint. It complexes with cyclindependent kinases, which then phosphorylate other cell cycle proteins (including Rb) to allow cell cycle progression. Cyclin D1 function is thought to be downregulated by the p53 gene through the mediation of p21. Thus, a cell cycle protein network exists, and an increase in cyclin D1 would be expected to drive the cell cycle, to increase cell proliferation and to increase the chance of additional oncogenic events.15,16,20,27–31 The IHC demonstration of cyclin D1 expression in most of the cases (95.1%) in the present study suggests that they may be primed for proliferation but that expression of p21 and/or other related proteins may have a restraining influence. The Rb gene is a prototypical tumour suppressor gene and encodes a 105–110 kDa nuclear phosphoprotein. Loss of Rb protein function is considered to be a key event in the development of a variety of human neoplasms. Rb gene alterations generally result either in the loss of Rb protein expression or in the formation of a truncated Rb protein that cannot enter the nucleus. Heterogeneous positive nuclear Rb staining generally indicates normal Rb function, and negative staining reflects functional loss of the Rb gene. Rb phosphorylation and formation of an active form at the G1–S transition is driven by Cdks, Cdk4 and Cdk6, in protein complexes with cyclin D1. Thus, Rb function correlates with the level of cyclin D1 and associated proteins (p15, p16, p18, Cdks).15,30–32 The presence or absence of normal Rb protein expression, as determined by IHC, has been found to be the most specific way to determine the Rb status of a given tumour. In the present study, however, there was no significant difference in the expression of Rb between the benign and malignant groups of carcinoid tumours. The bcl-2 oncogene was initially discovered at the breakpoint of the t(14;18) chromosomal translocation which is common in follicular lymphomas. This gene encodes an inner mitochondrial membrane protein of 26 kDa that protects cells from apoptosis. Recent studies have revealed a close relationship between aberrant bcl2 expression and tumour progression in a variety of human malignancies.13,33–36 Abnormal expression of bcl-2 has been reported to occur in dysplastic lesions of the gastrointestinal tract and to persist throughout neoplastic progression.37,38 Our results, however, suggest that
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abnormal expression is not of importance in the progression of gastrointestinal carcinoid tumours. Ki-67 reflects the proliferative activity and biological behaviour of tumours.5,14,35 In the present study the rate of Ki-67 positivity was higher in the malignant group than in the benign group (50% vs 0%) and, while the difference was not significant, one case with metastasis to the liver showed an extremely high Ki-67 positive rate. Thus, patients with a gastrointestinal carcinoid can be suspected of having distant metastases if the Ki-67 labelling index is high.
CONCLUSION In conclusion, the present study has demonstrated that abnormal expression of p21 and EC is common in malignant gastrointestinal carcinoid tumours and that their expression is correlated with advanced clinical stage. In addition, Ki-67 may be a biological marker of distant metastasis. The data suggest that p21, EC and Ki67 may have prognostic applications in carcinoid tumours, and this should be explored further in a larger study.
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Accepted for publication 16 October 2001
Immunohistochemical prognostic indicators of gastrointestinal carcinoid tumours M. Kawahara∗, M. Kammori∗, H. Kanauchi∗, C. Noguchi∗, S. Kuramoto∗, M. Kaminishi∗, H. Endo† and K. Takubo‡ Departments of ∗ Gastroenterological Surgery and † Pathology, University of Tokyo and ‡ Department of Clinical Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
Aims: The aim of this study was to determine whether expression of the oncoproteins p21, p53, E-cadherin (EC), cyclin D1, bcl-2 and Rb and the proliferation marker Ki-67 is predictive of malignant behaviour in gastrointestinal carcinoid tumours. Methods: Immunohistochemical (IHC) staining was performed on carcinoid tumours from 41 patients (31 rectal, eight gastrointestinal, two appendiceal lesions). The six tumours that had invaded deeply into the muscularis propria or beyond, had metastasized to regional lymph nodes or had metastasized to a distant site were classified as the malignant group, and the other 35 tumours formed the benign group. IHC expression was compared between the two groups, and the prognostic value of each marker was assessed. Results: Of the six tumours in the malignant group, 66.7% were p21 positive, 0% were p53 positive, 33.3% were EC positive, 100% were cyclin D1 positive, 33.3% were Rb positive, 16.7% were bcl-2 positive and 50% were Ki-67 positive. Of the 35 tumours in the benign group, 17.1% were p21 positive, 0% were p53 positive, 100% were EC positive, 94.3% were cyclin D1 positive, 8.6% were Rb positive, 17.1% were bcl-2 positive and 0% were Ki-67 positive. Conclusions: These data show that p53, cyclin D1, Rb, bcl-2 and Ki-67 staining does not correlate with malignant behaviour but that overexpression of p21 (P=0.02) and reduced staining of EC (P=0.005) do correlate with malignant behaviour. These two parameters may therefore be useful as prognostic indicators for gastrointestinal carcinoid tumours. 2002 Elsevier Science Ltd Key words: carcinoid tumour; immunohistochemistry; oncoprotein.
INTRODUCTION Carcinoid tumours are uncommon neoplasms that arise from neuroendocrine cells and occur most commonly in the gastrointestinal tract. They may produce a wide variety of biologically active agents. Although they may metastasize, their behaviour is very variable,1–3 and conventional classifications, including tumour, node and metastasis staging, are rather unsatisfactory because of the variation in survival within each entity. At present, little is known about the biological parameters that predict the disease course of carcinoid tumours.4–7 Treatment options for gastrointestinal carcinoid tumours include enterectomy with lymph node resection,8 local surgical excision9 and endoscopic resection.10,11 As
Correspondence to: Masaki Kawahara, MD, Department of Gastroenterological Surgery, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. Tel: +81-3-3815-5411; Fax: +813-5800-9734; E-mail: [email protected] 0748–7983/02/020140+07 $35.00/0
noted above, however, the behaviour of these tumours is variable, and criteria for selecting the most appropriate therapeutic approach have yet to be established. Identification of prognostic indicators that can be determined histologically might provide guidance for selecting optimal treatment of these tumours. The prognosis and choice of therapy for human neoplasms are determined by the stage of the disease and their likely behaviour. At present, their likely behaviour is estimated by routine histopathological examination of tissue sections obtained from the primary lesion. However, traditional histopathological findings are insufficient to identify patients with gastrointestinal neoplasms who are at risk of metastasis or recurrence.12 Molecular phenotyping has recently provided a useful extra dimension for characterization of biological potential, and it can be of assistance in predicting of clinical outcome. In this study, we assessed several oncoproteins that correlate with tumorigenesis in order to evaluate the malignant potential of gastrointestinal carcinoid tumours. 2002 Elsevier Science Ltd
IMMUNOHISTOCHEMISTRY OF GASTROINTESTINAL CARCINOID TUMOURS
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Table 1 Characteristics of the 41 patients with gastrointestinal carcinoids Characteristics Sex Location of tumour
Depth of invasion Lymph node metastasis Distant metastasis Vessel invasion Treatment
Number of patients Male Female Rectum Appendix Stomach, duodenum -sm pm+ − + − + − Surgical resection Endoscopic resection
27 14 31 2 8 38 3 3 38 1 40 4 37 38 3
-sm, mucosa or submucosa; pm-, tumour invasion to the muscularis propria or deeper layer; +, positive; −, negative; vessel invasion, lymphatic and/or venous invasion.
The objectives of the study were (a) to characterize the expression of p21, p53, E-cadherin (EC), cyclin D1, bcl-2, Rb, and Ki-67 in gastrointestinal carcinoid tumours by immunohistochemistry (IHC), (b) to investigate the significance of these oncoproteins by comparing IHC results in ‘malignant’ and ‘benign’ lesions and (c) to assess the prognostic value of expression of these markers.
MATERIALS AND METHODS Tissues and patient characteristics Archival tissues from 41 patients with gastrointestinal carcinoid tumours who had undergone surgical (n=38) or endoscopic (n=3) resection in our department or an affiliated hospital between January 1982 and December 1998 were used. Briefly, the patients’ mean age was 60 years (range: 39–86 years), and the male:female ratio was 27:14. Thirty-one patients had a rectal carcinoid, eight had a gastroduodenal carcinoid, and two had an appendiceal carcinoid (Table 1). In this series ‘malignant’ carcinoid tumours was defined as a carcinoid tumour with deep invasion into the muscularis propria or beyond, regional lymph node metastasis or distant haematogenous spread. Carcinoid tumours limited to the submucosal layer, without regional lymphatic or distant metastasis, were classified as benign.
IHC staining Sections of paraffin-embedded blocks from the carcinoid tumours were studied by IHC using monoclonal antibodies to p21 (1:20 dilution, Clone EA10; Oncogene Research Products, Cambridge, MA, USA), p53 (1:100 dilution, Clone DO-7; DACO, Glostrup, Denmark), EC
(1:50 dilution, Clone HECD-1, Takara Shuzo, Shiga, Japan), cyclin D1 (1:10 dilution, Clone DSC-6; PROGEN, Heidelberg, Germany), Rb (1:50 dilution, Clone 3H9; MBL, Nagoya, Japan), bcl-2 (1:50 dilution, Clone 124; DACO, Glostrup, Denmark) and Ki-67 (1:50 dilution, Clone Mib-1; IMMUNOTECH, Marseille, France), with the avidin–biotin complex (ABC) method. Briefly, 5 m thick sections were cut from the blocks, deparaffinized with xylene and dehydrated through graded concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. The sections were then treated with microwave radiation for 10 min for antigen retrieval, and, to block intrinsic antibody binding, they were reacted with normal serum (mouse IgG) for 10 min at room temperature. After the tissue sections had been exposed to primary antibodies overnight at 4°C, with appropriate negative and positive controls, they were reacted with biotinylated anti-mouse antibody (secondary antibody) for 10 min and with ABC for another 10 min, with intervening washes. Diaminobenzidine tetrahydrochloride was used as the final chromogen, and sections were counterstained with Mayer’s haematoxylin before mounting.
Specimen analysis IHC evaluation was conducted by two independent investigators (M.K. and K.T.), who scored the estimated percentage of tumour cells that exhibited nuclear, cytoplasmic and cell membrane staining. The percentage of immunoreactive tumour cells for each primary antibody used was evaluated. Positivity for each antibody was defined as reported previously.13–16 Tumour cells were considered strongly positive (2+) for p53 (nuclear
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Table 2 Category definitions for positive immunoreactivity Stain
p53 p21 bcl-2 EC CycD1 Ki-67 Rb
Positive
Negative
Strong
Intermediate
>50% >50% >50% >90% overexpression [10% [10% Overexpression
5–50% 5–50% 5–50% 10–90%
<5% <5% <5% <5% <10% <10% No expression
CycD1, cyclin D1; over- and no expression were defined by comparing with the internal control.
staining), p21 (nuclear staining), or bcl-2 (cytoplasmic staining) if >50% showed strong reactivity, intermediately positive (1+) if between 5% and 50% were immunoreactive and negative (−) if <5% were immunoreactive. EC (cytoplasmic and membrane staining) was regarded as strongly positive (2+) if >90% of tumour cells showed stronger expression than the surrounding non-neoplastic tissue, intermediately positive (1+) if 10–90% were immunoreactive and negative (−) if <10% were immunoreactive. Tumour cells were considered positive for cyclin D1 (nuclear staining) and Ki-67 (nuclear staining) when [10% of the cells were immunoreactive. Staining for Rb was scored as Rb negative if none of the tumour cells exhibited nuclear staining and the Rb-positive cells in background tissue showed adequate nuclear staining as a positive internal control (Table 2). The IHC analysis was performed blind, without knowledge of the clinical data.
Statistical analysis Logistic regression was used for univariate and multivariate categorical analysis of IHC staining. P<0.05 was considered significant.
RESULTS Pathological and clinical characteristics Patient characteristics and the histopathological findings in the 41 tumours are listed in Table 1. Of the 41 tumours, three showed deep invasion, three had metastasized to lymph nodes and one had metastasized to the liver. A total of six tumours with metastasis or deep invasion were classified in the malignant group (Table 3).
IHC staining for p53, cyclin D1, Rb and bcl-2 No tumours showed positive staining for p53 protein. Cyclin D1 (2+) protein was detected in most of the
tumours, 100% of the malignant group and 94.3% of the benign group, but staining was generally stronger in the former (Fig. 1). Rb expression was variable: 33.3% (two of six) were positive in the malignant group and 8.6% (three of 35) were positive in the benign group. Bcl-2 was little different between the two groups: 16.7% (one of six) were intermediately positive (1+) in the malignant group and 17.1% (six of 35) intermediate positive (1+) in the benign group. No tumours showed strong positive staining for bcl-2 protein. Accordingly, there was no significant correlation between p53, cyclin D1, Rb or bcl-2 expression and the malignant behaviour of the carcinoid tumours (Table 4).
IHC staining of p21, EC and Ki-67 Immunoreactivity, indicative of p21 protein overexpression (1+) (Fig. 2), was found in four of the six tumours (66.7%) in the malignant group, vs six of the 35 tumours (17.1%) in the control group. The difference was significant (P<0.05) with 5% positive expression as the cut-off point.14,16 Two of the six tumours (33.3%) in the malignant group showed strongly positive (2+) cytoplasmic and membrane staining for EC (Fig. 3) and four (66.7%) were negative (−), whereas 100% of the tumours in the benign group showed strong positive (2+) expression (Fig. 4). We therefore employed 90% positive expression as the cut-off point for EC. This difference was also statistically significant (P<0.05), and the carcinoid cells in the benign group showed much stronger expression than those in the malignant group. Tumour cell nuclei were considered positive for Ki67 antigen when they showed dark brown granular staining. More than threshold staining was evident in 50.0% (three of six) of the malignant group but in none of the tumours in the benign group. The difference, however, did not reach statistial significance. One case of gastroduodenal carcinoid with liver metastases (case 4 in Table 3) had a high Ki-67-positive rate (Fig. 5), and
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Table 3 Summary of details for the six patients with malignant carcinoids Number
Age (years)
Sex
Location
Size (mm)
Depth
Lymph node metastasis
Distant metastasis
Outcome
46 61 69 51 86 77
F M M M M M
R R R D T A
15 9 8 22 62 60
pm sm sm pm ss pm
− + + + − +
− − − + − −
Alive Alive Alive ED NED NED
1 2 3 4 5 6
F, female; M, male; R, rectum; D, duodenum; T, transverse colon; A, appendix; pm, muscularis propria; sm, submucosa; ss, subserosa; +, positive; −, negative; ED, dead with evidence of disease; NED, dead with no evidence of disease.
Figure 1 Cyclin D1 immunostain of a carcinoid tumour of the rectum demonstrating strong immunoreactivity of tumour in comparison with the adjacent non-neoplastic glandular cells (×100).
Figure 2 p21 immunostain showing positive immunoreactivity of tumour cells of a duodenal carcinoid tumour with a malignant phenotype (×200).
Table 4 Summary of data for immunostaining of the carcinoid tumours Stain
p53 p21 EC CycD1 Ki-67 bcl-2 Rb
Malignant group (n=6)
Benign group (n=35)
Positive
Negative
Positive
Negative
P value
0% 66.7% 33.3% 100% 50.0% 16.7% 33.3%
100% 33.3% 66.7% 0% 50.0% 83.7% 66.7%
0% 17.1% 100% 94.3% 0% 17.1% 8.6%
100% 82.9% 0% 5.7% 100% 82.9% 91.4%
1.00 0.02 0.005 0.94 0.09 0.88 0.15
CycD1, cyclin D1.
the patient was treated by distal gastrectomy and partial hepatectomy, but died of recurrent liver metastases. There was no interaction between parameters when the categorical data for IHC expression were subjected to multivariate logistic analysis.
DISCUSSION Carcinoid tumours are generally characterized by slower evolution and lower grade malignant behaviour than carcinomas. However, it is well known that carcinoid
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Figure 3 EC immunostain showing negative reactivity of tumour cells of a rectal carcinoid tumour with a malignant phenotype (×100).
Figure 4 EC immunostain showing positive reactivity of tumour cells of a rectal carcinoid tumour with a benign phenotype (×100).
Figure 5 Ki-67 immunostain demonstrating strong nuclear positivity of tumour cells of a gastric carcinoid tumour (×200).
tumours may metastasize, and optimal treatment is controversial because of the difficulty in identifying features of aggressive behaviour. A number of parameters
M. KAWAHARA ET AL. have been proposed for predicting the clinical behaviour and outcome of carcinoid tumours, and tumour size, depth of invasion and histological appearance have been considered the most reliable.8–10 Nevertheless, the behaviour of this group of tumours still remains unpredictable, and this sometimes leads to difficulty in selecting the most appropriate treatment for an individual patient. More reliable prognostic indicators for carcinoid tumours are therefore required. Recent advances in molecular biology and in our comprehension of carcinogenesis and tumour development have provided new tools with which to determine the metastatic potential of human malignancies. The p53 gene encodes a 53 kDa nuclear phosphoprotein that is involved in the regulation of cell proliferation and apoptosis. Mutation of the p53 gene results in neoplastic transformation in a wide variety of human organs and tissues, as typified by carcinoma of the colon.13–18 However, we found no significant correlation between p53 overexpression and malignant behaviour in gastrointestinal carcinoid tumours in the present study. This is in agreement with the findings in a previous report on colorectal carcinoid tumours.19 The oncoprotein for p21 is a p53 downstream protein and plays a key role in the p53 suppressor function that controls the G1–S cell cycle checkpoint. Since the p21 gene is transcriptionally activated by wild-type p53, alteration of p53 function may result in loss of p21 expression, and this may be one of the mechanisms by which altered p53 influences tumour progression.14,16–18,20 Conversely, overexpression of p21 is considered to lead to inhibition of cellular growth and proliferation.14,17,20 Although our study showed no p53 overexpression, IHC staining of p21 protein, an indicator of overexpression, was significantly more common in the malignant group than in the benign group. This was unexpected in view of the negative p53 staining, but it has been demonstrated that p21 expression can also be mediated through p53-independent pathways.14,21,22 EC, a member of the cadherin family of cell surface glycoproteins, is a calcium-dependent cell-to-cell adhesion molecule and invasion suppressor molecule that is found in epithelial tissues. It effects its adhesive function via the homotopic interactions of five extracellular repeated domains, with its intracellular portion complexing with several distinct undercoat proteins, including members of the catenin family. Loss of EC function is closely related to malignant behaviour, which is characterized by dedifferentiation, invasion, metastasis and overproliferation.12,13,22–26 We observed overexpression of EC in all the benign carcinoid tumours in this series, and it may be possible to use EC overexpression as an IHC marker for the diagnosis of carcinoid tumours. However, this needs to be studied in many more cases. We also observed a significant correlation between markedly reduced expression of
IMMUNOHISTOCHEMISTRY OF GASTROINTESTINAL CARCINOID TUMOURS EC and degree of malignancy, suggesting a relationship between loss of cell adhesion and progression of carcinoid tumours. In other words, EC overexpression appears to be a favourable prognostic indicator for carcinoid tumours. To our knowledge this is the first report of an association between EC expression, as determined by IHC, and the prognosis of carcinoid tumours. Cyclin D1 is a 45 kDa cell cycle regulatory protein that is transcribed from the CCND1 gene at 11q13 and acts at the late G1 checkpoint. It complexes with cyclindependent kinases, which then phosphorylate other cell cycle proteins (including Rb) to allow cell cycle progression. Cyclin D1 function is thought to be downregulated by the p53 gene through the mediation of p21. Thus, a cell cycle protein network exists, and an increase in cyclin D1 would be expected to drive the cell cycle, to increase cell proliferation and to increase the chance of additional oncogenic events.15,16,20,27–31 The IHC demonstration of cyclin D1 expression in most of the cases (95.1%) in the present study suggests that they may be primed for proliferation but that expression of p21 and/or other related proteins may have a restraining influence. The Rb gene is a prototypical tumour suppressor gene and encodes a 105–110 kDa nuclear phosphoprotein. Loss of Rb protein function is considered to be a key event in the development of a variety of human neoplasms. Rb gene alterations generally result either in the loss of Rb protein expression or in the formation of a truncated Rb protein that cannot enter the nucleus. Heterogeneous positive nuclear Rb staining generally indicates normal Rb function, and negative staining reflects functional loss of the Rb gene. Rb phosphorylation and formation of an active form at the G1–S transition is driven by Cdks, Cdk4 and Cdk6, in protein complexes with cyclin D1. Thus, Rb function correlates with the level of cyclin D1 and associated proteins (p15, p16, p18, Cdks).15,30–32 The presence or absence of normal Rb protein expression, as determined by IHC, has been found to be the most specific way to determine the Rb status of a given tumour. In the present study, however, there was no significant difference in the expression of Rb between the benign and malignant groups of carcinoid tumours. The bcl-2 oncogene was initially discovered at the breakpoint of the t(14;18) chromosomal translocation which is common in follicular lymphomas. This gene encodes an inner mitochondrial membrane protein of 26 kDa that protects cells from apoptosis. Recent studies have revealed a close relationship between aberrant bcl2 expression and tumour progression in a variety of human malignancies.13,33–36 Abnormal expression of bcl-2 has been reported to occur in dysplastic lesions of the gastrointestinal tract and to persist throughout neoplastic progression.37,38 Our results, however, suggest that
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abnormal expression is not of importance in the progression of gastrointestinal carcinoid tumours. Ki-67 reflects the proliferative activity and biological behaviour of tumours.5,14,35 In the present study the rate of Ki-67 positivity was higher in the malignant group than in the benign group (50% vs 0%) and, while the difference was not significant, one case with metastasis to the liver showed an extremely high Ki-67 positive rate. Thus, patients with a gastrointestinal carcinoid can be suspected of having distant metastases if the Ki-67 labelling index is high.
CONCLUSION In conclusion, the present study has demonstrated that abnormal expression of p21 and EC is common in malignant gastrointestinal carcinoid tumours and that their expression is correlated with advanced clinical stage. In addition, Ki-67 may be a biological marker of distant metastasis. The data suggest that p21, EC and Ki67 may have prognostic applications in carcinoid tumours, and this should be explored further in a larger study.
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Accepted for publication 16 October 2001