Goblet Cell Carcinoids of the Appendix. Immunophenotype and Ultrastructural Study

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1 Goblet Cell Carcinoids of the Appendix Immunophenotype and Ultrastructural Study R. Kanthan, MBBS, MS, FRCS, FRCPC; A. Saxena, MBBS, MD, FRCPC, FCAP; S. C. Kanthan, MBBS, FRCS, FRCSC Background. Goblet cell carcinoids of the appendix are rare neoplasms with uncertain biological behavior. Objective. The aims of our study were to evaluate the immunophenotype of this neoplasm with cell cycle/cell proliferation markers and to understand their histogenesis with ultrastructural analysis using conventional carcinoids as a frame of reference. Methods. Clinical data and archival pathologic material of all goblet cell carcinoids of the appendix recorded by the Saskatchewan Cancer Registry between 1970 and 1998 were reviewed and evaluated by light microscopy, histochemistry, immunohistochemistry, and electron microscopy. Results. Seven cases of goblet cell carcinoids were identified among 110 cases of conventional carcinoids of the appendix. Histopathology revealed widespread infiltration of the periappendiceal fat in all cases, with extensive perineural invasion. The cells stained strongly positive for mucicarmine, periodic acid Schiff, periodic acid Schiff diastase, Alcian blue, cytokeratin, and carcinoembryonic antigen. Most cases were positive for synaptophysin. Increased expression of cell proliferation markers and cell cycle markers was observed. Expression of p53 was strong in one case. Electron microscopy demonstrated the presence of mucinous vacuoles of varying sizes and occasional membrane-bound neuroendocrine granules. Conclusions. Goblet cell carcinoids of the appendix arise from a pluripotent cell with divergent neuroendocrine and mucinous differentiation. These neoplasms are widely invasive; they demonstrate a high cellular proliferation rate and dysregulation of the cell cycle with up-regulation of cyclin D1 and p21, and down-regulation of p16. Complete removal of the tumor is recommended because of the unpredictable biological behavior of this tumor, which includes delayed local recurrences and lung metastases. (Arch Pathol Lab Med. 2001;125: ) Goblet cell carcinoid of the appendix as a distinct entity was first described in Dual endocrine and glandular differentiation has led to confusion in the nomenclature (adenocarcinoid, 2 crypt cell carcinoma, 3 and mucinous carcinoid 4 ) and management of such lesions. Goblet cell carcinoids are uncommon neoplasms, and their exact biological behavior is uncertain. In the past, these tumors were alleged to be associated with the same indolent clinical course as conventional carcinoids. However, more recent studies indicate aggressive behavior. 5 7 The aims of our study were to evaluate the histomorphologic and immunohistochemical features of this neoplasm, with special reference to cell cycle and proliferation markers, and to delineate their histogenesis with added ultrastructural analysis. METHODS A computer-based data search of carcinoids of the appendix at the Saskatchewan Cancer Registry (Saskatoon, Saskatchewan) Accepted for publication October 10, From the Departments of Pathology (Drs R. Kanthan and Saxena) and Surgery (Dr S. C. Kanthan), Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan. Presented in part at a meeting of the Canadian Association of General Surgeons, The Royal College of Physicians and Surgeons of Canada, Montreal, Ontario, September 24, Reprints: Rani Kanthan, MBBS, MS, Department of Pathology, Royal University Hospital, 103 Hospital Dr, Saskatoon, Saskatchewan, Canada S7 OW8. was conducted for the years 1970 through Clinical data and archival material of all goblet cell carcinoids of the appendix were reviewed. Goblet cell carcinoids were defined as tumors composed primarily of nests of mucin-filled goblet cells surrounded by stroma. Goblet cell carcinoids with adjacent adenocarcinoma or areas of solid growth were excluded. A detailed morphologic analysis of hematoxylin-eosin stained, 3- m-thick slides obtained from formalin-fixed, paraffin-embedded tissue blocks was performed. Representative sections were stained with periodic acid Schiff (PAS), PAS diastase, Alcian blue, mucicarmine, Fontana-Masson, and Churukian Schenk. Sections were stained by routine immunohistochemical methods, including antigen-retrieval and avidin-biotin complex techniques. The antibodies used in this study are listed in Table 1. Tissue blocks for electron microscopy were taken from the formalin-fixed specimens and postfixed in 3% glutaraldehyde. In 3 cases, material was obtained from the paraffin-fixed blocks. These samples were then processed in the traditional fashion. RESULTS Seven cases of goblet cell carcinoids were identified among 110 cases of conventional carcinoids of the appendix. Table 2 details the relevant clinicopathologic profiles of these cases. The clinical presentation in all 7 cases was an acute abdomen with florid acute appendicitis. All cases had a right hemicolectomy as a primary or interval secondary procedure. Three patients died with carcinomas of the lung (pathologically confirmed to be metastatic in 1 case), with a mean survival of 8 years. Late local recurrence was observed in 1 case. The mean follow-up of the 386 Arch Pathol Lab Med Vol 125, March 2001 Goblet Cell Carcinoids of Appendix Kanthan et al

2 Table 1. Antibodies Used in This Study Antibody Clone Dilution Source Ki-67 MIB-1 1:50 Immunotech, Marseilles, France Bcl-2 Bcl-2/100/D5 Medical Systems Inc, Tucson, Ariz c-erb-b2 Polyclonal 1:300 Dako Corporation, Carpinteria, Calif p53 DO7 1:50 ovocastra Laboratories Ltd, ewcastle Upon Tyne, United Kingdom CK CAM5.2 1:20 Becton Dickinson, Mountain View, Calif CEA PCA p16 p21 Cyclin D1 CGA SYP TF3H8-1 PC10 IK4 EA10 P2D11F11 LK2H10 Polyclonal 1:250 1:5 Predlute Pharmingen, Santa Cruz, Calif Oncogene Research Products, Calbiochem, Cambridge, Mass * CK indicates cytokeratin; CEA, carcinoembryonic antigen; PCA, proliferating cell nuclear antigen; CGA, chromogranin A; and SYP, synaptophysin. Table 2. Clinicopathologic Profile of 7 Cases of Goblet Cell Carcinoid of the Appendix* Case Designation Age, y/sex Presentation Clinical Course Outcome, y A 82/F Perforated appendicitis Appendectomy, interval hemicolectomy ED, 1.3 B 34/M Acute appendicitis Appendectomy, interval hemicolectomy ED, 2.5 C 61/M Perforated appendicitis Appendectomy (1975), carcinoma DOD, 19.2 of lung (1991), right hemi- colectomy (1993) D 62/M Acute appendicitis Appendectomy, interval hemicolectomy ED, 2.3 E 68/F Perforated appendicitis Appendectomy (1980), stage III DOC, 5.4 F 61/M Acute appendicitis, bowel obstruction, diffuse pneumonia carcinoma of lung (1985) Appendectomy (1986), right hemicolectomy (1989), carcinoma of lung metastatic (1990) DOD, 4.6 G 50/M Obstructing lesion in appendix Right hemicolectomy ED, 1.2 * ED indicates no evidence of disease; DOD, died of disease; and DOC, died of other causes. Table 3. Immunohistochemical Staining in 7 Cases of Goblet Cell Carcinoid of the Appendix* Case Designation p53 p16 p21 Cyclin D1 Ki-67 PCA CGA SY CEA A B C D E F G 40S 60M 60M 30W * umeric values represent the percentage of cells being stained. indicates negative staining; S, strong staining; M, moderate staining; W, weak staining; PCA, proliferating cell nuclear antigen; CGA, chromagranin A; SYP, synaptophysin; and CEA, carcinoembryonic antigen. 30S 10M 30S 40S 20M 10M 50M 30M 95S 85S 95S remaining 4 cases showed no evidence of disease at 18 months. Histopathology revealed the presence of acute appendicitis with widespread neoplastic infiltration of the periappendiceal fat in all cases; extensive perineural invasion was also evident (Figure 1). The tumor was composed of adenomatous groups of goblet cells containing predominantly vacuolated cytoplasm with basal round to crescentic nuclei containing a single small nucleolus. Many cells resembled the signet ring type. Less conspicuous were cells with pink granular cytoplasm and round nuclei (Figure 2). The vacuolated cells stained strongly with mucicarmine and Alcian blue, and those with eosinophilic granular cytoplasm stained with PAS after diastase digestion. Reactivity with Fontana-Masson and Churukian Schenk stains was predominantly negative, but occasional cells were positive. Immunohistochemical staining of the tumor cells revealed strong positive staining with antibodies to cytokeratin and carcinoembryonic antigen. egative staining Arch Pathol Lab Med Vol 125, March 2001 Goblet Cell Carcinoids of Appendix Kanthan et al 387

3 Figure 1. a and b, Acute appendicitis with a diffusely infiltrating goblet cell neoplasm (hematoxylin-eosin, original magnification 50 [a] and 125 [b]). c, Perineural invasion is evident (hematoxylin-eosin, original magnification 500). Figure 2. Morphology of goblet cell carcinoids at (a) high power (hematoxylin-eosin, original magnification 750) and (b) medium power (hematoxylin-eosin, original magnification 250). patterns were observed with antibodies to Bcl-2 and c-erb- B2. Staining with neuroendocrine markers was inconsistent. Many cells were negative for chromogranin A and diffusely positive for synaptophysin. Cell proliferation markers showed an increased expression of Ki-67 (30% to 60%) and proliferating cell nuclear antigen (60% to 90%). Overexpression of cyclin D1 (30% to 70%) and p21 (40% to 60%) with negative staining for p16 were also observed. One case strongly expressed p53 (80%). Results of immunohistochemical studies are summarized in Table 3. The conventional carcinoids showed no such trends of overexpression of cell proliferation and/or cell cycle markers. Ultrastructurally, tumor cells showed the presence of mucinous vacuoles of varying sizes with occasional membrane-bound, electron-dense granules in all 7 cases. The latter were few in number and extremely difficult to locate (Figure 3) in many of the cases, in contrast to the conventional carcinoids. COMMET Carcinoid tumors are unusual neoplasms. The most frequent site for carcinoids is the gastrointestinal tract (73.7%), with the appendix accounting for 18.9% of the cases. 8,9 Recent data suggest that after adenocarcinoma, carcinoids are the second most common primary malignant tumor arising in the appendix. 10 The estimated prevalence ranges from 0.32% to 0.8% of patients undergoing appendectomy. 11 In 1969, Gagne et al 12 described a series of 3 appendiceal tumors that had both argentaffin- and mucin-secreting cells. Subbuswamy et al 1 reported on 12 similar cases and labeled them goblet cell carcinoids. Various names have since been proposed for these carcinoids with dual differentiation, including crypt cell carcinoma, 3 mucinous carcinoid, 4 and adenocarcinoid. 2 Adenocarcinoid seems to be the favored term in recent reports. Histologic identification of these tumors is fairly straightforward in most cases. They are recognized as 388 Arch Pathol Lab Med Vol 125, March 2001 Goblet Cell Carcinoids of Appendix Kanthan et al

4 Figure 3. Electron microscopic examination reveals cells with abundant mucinous vacuoles and few electron-dense granules (arrow) (original magnification ). neoplasms primarily infiltrating the submucosa and the muscular wall in a circumferential fashion, sparing the mucosa. In some of our cases, the goblet tumor cells were typically poorly defined among the relatively abundant stroma and/or smooth muscle, resulting in difficulty in their identification, particularly amid the background of acutely florid inflammation of the appendix. The glands lining these compact clusters of cells contained intracytoplasmic mucin and were mucicarmine and Alcian blue positive with no central lumen. o mucinous lakes were seen in any of our cases. Occasional foci of fused goblet cell nests with signet ring cells were also seen. Some cells had PAS diastase positive eosinophilic granular cytoplasm. The nuclei were crescent-shaped or round and basally oriented, often with a single small nucleolus. Diffuse infiltration into the periappendiceal fat and perineural invasion was seen in most cases. This feature needs to be carefully evaluated in the assessment of microscopically negative resection margins in these cases, as these neoplasms do not form discrete masses. All tumors in our cases showed endocrine differentiation (immunohistochemical and/or ultrastructural), although the number of these cells was uniformly low. All cases were positive for carcinoembryonic antigen and cytokeratin. o serum values of carcinoembryonic antigen were available, although this might be considered in the clinical follow-up of these patients. In none of our cases was the diagnosis suspected preoperatively or established intraoperatively. The clinical presentation in all cases was one of an acute abdomen suggestive of appendicitis, resulting in appendectomy as a primary procedure. One case presented with an obstructing appendiceal lesion, resulting in a right hemicolectomy as a primary procedure. In most cases, the appendix had acute florid inflammation, and 3 cases had identifiable perforation and abscess. A male predominance of 5:2 was seen in our cases. This finding is in contrast to results reported in other studies 8 10 and is perhaps related to a different population sampling. The age at presentation ranged from 34 to 82 years, with a mean of 59 years. The clinical course was variable. Pathologically proven death caused by local metastases was established in one case, and distant disease to the lung was recorded in another. Survival times from the time of diagnosis to death were 4.6, 5.4, and 19.2 years in the 3 patients who died. This wide range confirms previous reports of the unpredictable malignant behavior of these tumors. 5 7 Our recommendation in these cases includes life-long clinical follow-up for all patients. The unpredictable behavior of this tumor is probably related to its unique biology. Our study supports an increased proliferation rate, despite a lack of visible mitoses. It is our opinion that there is a large reserve G0/G1 pool of pluripotent cells that remain noninvasive until further genetic instability is acquired by the cell. Dysregulation of the cell cycle may be one pathway to this genomic alteration, as supported by overexpression of cyclin D1 and p21. We believe this to be a p53-independent pathway, as immunohistochemical methods detected p53 in only one of our cases. K-ras mutations and p53 accumulation by immunohistochemistry have not been detected in goblet cell carcinoids. 13 However, single cases with such expression have been designated as aggressive phenotypes 14 ; we suggest caution in such interpretation unless supported by molecular genetic analysis. The histogenesis of these tumors remains a confounding problem Ultrastructural analysis reveals the presence of goblet cells with occasional electron-dense granules between pale mucinous globules of varying sizes. In our opinion, such divergent dual differentiation probably arises from a pluripotent intestinal stem cell with disorderly differentiation. We believe these neoplasms belong to the class of amphicrine tumors with both exocrine and endocrine features. 18 In summary, we believe that goblet cell carcinoids of the appendix are amphicrine tumors arising from a pluripotent cell with divergent neuroendocrine and mucinous differentiation. These neoplasms have a high cellular proliferation rate and dysregulation of the cell cycle with upregulation of cyclin D1 and p21, and down-regulation of p16. The exact understanding of the tumor biology of these neoplasms is still in its evolution. Although appendiceal carcinoids are potentially lethal neoplasms, tumor eradication is usually feasible and effective. Owing to the pattern of diffuse perineural and appendiceal wall infiltration, we recommend complete removal (with microscopically negative margins) of the goblet cell variety of such carcinoids of the appendix. We thank Todd Reichert and Bob van den Beuken for all their help in the preparation of the images for this article. References 1. Subbuswamy SG, Gibbs M, Ross CF, Morson BC. Goblet cell carcinoid of the appendix. Cancer. 1974;34: Warkel RL, Cooper PH, Helwig EB. Adenocarcinoid, a mucin producing carcinoid tumor of the appendix. Cancer. 1978;42: Gallegos C, Milroy C, Linchai IP, Boulos PB. Crypt cell carcinoma of the appendix. Eur J Surg Oncol. 1992;18: Klein HZ. Mucinous carcinoid tumor of the vermiform appendix. Cancer. 1974;33: Arch Pathol Lab Med Vol 125, March 2001 Goblet Cell Carcinoids of Appendix Kanthan et al 389

5 5. Park K, Blessing K, Kerr K, Chetty U, Gilmour H. Goblet cell carcinoid of the appendix. Gut. 1990;31: Deans GT, Spence RA. eoplastic lesions of the appendix. Br J Surg. 1995; 82: Butler TA, Houshiar A, Lin F, Wilson SE. Goblet cell carcinoid of the appendix. Am J Surg. 1994;168: Modlin IM, Sandor A. An analysis of 8305 cases of carcinoid tumors. Cancer. 1997;79: Lauffer JM, Zhang T, Modlin IM. Review article: current status of Gastrointestinal carcinoids. Aliment Pharmacol Ther. 1999;13: Sandor A, Modlin IM. A retrospective analysis of 1570 appendiceal carcinoids. Am J Gastroenterol. 1998;93: Gouzi JL, Laigneau P, Delalande JP, et al. Indications for right hemicolectomy in carcinoid tumors of the appendix: the French Association for Surgical Research. Surg Gynaecol Obstet. 1993;176: Gagne F, Fortin P, Dufour V, Delage C. Tumeurs de l appendice associant des caracteres histiologiques de carcinoide et d adenocarcinome. Ann Anat Pathol. 1969;14: Capella C, la Rosa S, Ucella S, Billo P, Cornaggia M. Mixed endocrineexocrine tumors of the gastrointestinal tract. Semin Diagn Pathol. 2000;17: Horiuchi S, Endo T, Shimoji H, et al. Goblet cell carcinoid of the appendix: endoscopically diagnosed and examined with p53 immunostaining. J Gastroenterol. 1998;33: Goddard MJ, Lonsdale R. The histogenesis of appendiceal carcinoid tumors. Histopathology. 1992;20: Cooper P, Warkel R. Ultrastructure of the goblet cell type of adenocarcinoid of the appendix. Cancer. 1978;42: Anderson H, Somerville JE, Johnston CF, Hayes DM, Buchanan KD, Sloan JM. Appendiceal goblet cell carcinoids: a clinicopathological and immunohistochemical study. Histopathology. 1991;18: Mandoky L. Amphicrine tumor. Pathol Oncol Res. 1999;3: Arch Pathol Lab Med Vol 125, March 2001 Goblet Cell Carcinoids of Appendix Kanthan et al