Appendiceal Mucosal Schwann Cell Proliferation: A Putative Histologic Marker of Appendiceal Diverticular Disease

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1 494795IJSXXX / International Journal of Surgical PathologyStockl et al research-article2013 Original Article Appendiceal Mucosal Schwann Cell Proliferation: A Putative Histologic Marker of Appendiceal Diverticular Disease International Journal of Surgical Pathology 21(6) The Author(s) 2013 Reprints and permissions: sagepub.com/journalspermissions.nav DOI: / ijs.sagepub.com Thomas Stockl, MD, MPH 1, Jeffrey S. Ross, MD 2, Otto Walter, MD 1, Karen Dresser, BS 1 and Hwajeong Lee, MD 1,2 Abstract Recognition of an appendiceal diverticulum is important because of its association with an appendiceal neoplasm. The incidence of mucosal Schwann cell proliferation in 24 cases of appendiceal diverticular disease, 17 serrated polyps, 4 cases of mucosal hyperplasia, and 45 normal appendices was determined. Ten (42%) of 24 cases with diverticula, 2 (50%) of 4 cases of mucosal hyperplasia with concurrent surface low-grade dysplasia, and 9 (20%) of 45 cases of normal appendices showed mucosal Schwann cell proliferation. It was not seen within the 17 cases of serrated polyps. Mucosal Schwann cell proliferation is common in appendiceal diverticular disease and may serve as a histologic marker for the presence of an appendiceal diverticulum. Thus, when routine histologic sections of a removed appendix demonstrate Schwann cell proliferation, further examination of the specimen may detect possible coexisting diverticular disease, which in turn may be associated with appendiceal neoplasms and epithelial dysplasia. Keywords neural proliferation, Schwann cell, appendiceal diverticulum, intramucosal neuroma, serrated polyp Introduction Appendiceal diverticular disease is rare, with a reported incidence of 0.004% to 2.1% from appendectomies and 0.2% to 0.66% from autopsies. 1,2 In routine appendectomy specimens, it is difficult to identify an appendiceal diverticulum on gross examination because the size of the diverticulum is usually small. 1 In addition, the microscopic detection of an appendiceal diverticulum may be hampered by superimposed acute inflammation or accompanying mucosal architectural distortion. 3 An appendiceal diverticulum may be complicated by chronic pain, acute inflammation, and perforation. 1,4 In addition, it has been reported that 42% of low-grade appendiceal mucinous neoplasms are associated with appendiceal diverticular disease. 2 Moreover, given the findings in a recent retrospective study showing that 48% of the appendectomies with diverticular disease harbored a coexisting primary appendiceal neoplasm, including adenocarcinomas and neuroendocrine tumors proximal to the diverticula, investigators have recommended that a thorough examination of all appendectomy specimens with diverticula be conducted to exclude concomitant neoplastic disease. 4 Furthermore, a ruptured appendiceal diverticulum may simulate a low-grade appendiceal mucinous neoplasm, creating a potential diagnostic pitfall for the examining pathologist. 3 However, despite this importance of the correct identification of appendiceal diverticular disease, the histologic features associated with appendiceal diverticular disease are not well characterized in the published literature. Benign neural proliferation of the appendix is one of the lesser-known histologic findings associated with an appendiceal diverticulum. 2,3 Appendiceal neural proliferation has been described in the submucosa and mucosa of the appendix, with reported incidence ranging from 1.7% to 82% It has been referred to as neuroma, neurogenous hyperplasia, neurogenic appendicitis, neuromatosis, traumatic neuroma, fibrous obliteration, axial neuroma, neurogenic appendicopathy, neuroimmune appendicitis, and appendiceal fibrosis Some authors have divided 1 University of Massachusetts, Worcester, MA, USA 2 Albany Medical College, Albany, NY, USA Corresponding Author: Hwajeong Lee, Pathology and Laboratory Medicine, 47 New Scotland Avenue MC 81, Albany, NY 12208, USA. LeeH5@mail.amc.edu

2 604 International Journal of Surgical Pathology 21(6) Table 1. Intramucosal Schwann Cell Proliferation (SP) of the Appendix in the Literature. Reference Case SP Intramucosal SP Confirmatory Study Höfler et al, 13 Höfler 16 > % 10.4% Sub P, VIP, NSE Auböck and Ratzenhofer Unknown 16% Ultrastructure Stanley et al % 10% S-100, NSE, 5-HT, SOM Olsen and Holck % 6.2% a S-100 Naik % 33% b PTAH Gűller et al % 8.6% N/A Franke et al % 81% c S-100 Abbreviations: Sub P, substance P; VIP, vasoactive interstitial polypeptide; NSE, neuron-specific enolase; 5-HT, serotonin; SOM, somatostatin; N/A, not applicable; PTAH, phosphotungstic acid hematoxylin. a Cases associated with secondary mucosal hyperplasia. b Referred as moderate to severe mucosal neural proliferation. c Referred as mucosa type neurogenic appendicopathy. the proliferations into central obliterative type, nodular type, and intramucosal type according to the histologic growth pattern and location, of which the intramucosal type is less common. 7,9,11,13,15-17 The lesional neural cells have been uniformly S-100 positive Schwann cells and neuroendocrine cells with neurosecretory granules (Table 1). 11,15 The presence of these neural proliferations in published studies of routine appendectomy specimens have been attributed to long-term sequelae of acute appendicitis, chronic irritation, and intermittent obstruction. 5,8 To date, a detailed study of appendiceal neural and Schwann cell proliferation in appendiceal diverticular disease has not been reported, and the hypothesis that appendiceal mucosal Schwann cell proliferation may be a characteristic histologic finding of appendiceal diverticular disease has not been validated. In this study, we performed a retrospective routine histologic review of appendiceal diverticular diseases to evaluate mucosal Schwann cell proliferation and to determine its incidence and diagnostic significance. In comparison, we retrieved appendices with variable mucosal expansions and architectural distortions, including polyps and mucosal hyperplasia, and studied mucosal Schwann cell proliferation in these lesions. The findings were compared with those found in normal appendices. In addition, the presence of Schwann cells in the mucosa of the appendix was confirmed in all cases by immunohistochemistry. Materials and Methods The study was approved by the University of Massachusetts institutional review board. A retrospective search for appendectomy and colectomy cases with appendiceal lesions was performed from 2000 to Cases of lowgrade appendiceal mucinous neoplasm and acute appendicitis without mucosal expansion and diverticula were excluded. Finally, 45 cases with available tissue blocks were retrieved, which included the following: 24 cases of appendiceal diverticular disease (including 1 case with a concomitant serrated polyp), 17 appendiceal serrated polyps, and 4 cases of mucosal hyperplasia. Mucosal hyperplasia was defined as prominent hyperplastic change with increased mucin-containing cells, which is limited in the upper half of the mucosa, with no distinct villiform change or back-to-back crypts. We used 45 cases of normal appendices as negative controls. Hematoxylin and Eosin (H&E) Section Evaluation H&E slides were reviewed for the distribution, location, and density of intramucosal spindle cells. Spindle cells in the submucosa and muscularis were not evaluated. Distribution was designated as follows: focal, involving less than 10% of the mucosa; patchy, involving 10% to 75% of the mucosa; and diffuse, involving more than 75% of the mucosa. The location was either base, mid, apical, or full thickness. The density of the proliferation was determined as either loose (proliferation in a myxoid and pale stroma mixed with discernable inflammatory cells, including eosinophils, of the lamina propria) or dense (no intervening inflammatory cells of the lamina propria between the spindle cells with vague nodular growth pattern). Secondary changes such as prominent crypt architectural distortion and epithelial hyperplastic change were documented. Among the 45 normal appendices, any cases with prominent spindle cell proliferation in the lamina propria, associated with or without crypt architectural distortion, were submitted for immunoperoxidase staining. Immunohistochemical Stain Representative 4-µm tissue sections from paraffinembedded tissue blocks were stained for S-100 (Dako polyclonal rabbit, 1:1600 citrate antibody retrieval), epithelial

3 Stockl et al 605 membrane antigen (EMA; Dako monoclonal mouse clone E29, 1:25 citrate antibody retrieval), neurofilament protein (NFP; Dako monoclonal mouse clone 2F11, 1:400 proteinase K digestion), and claudin-1 (LSBio monoclonal mouse clone 1C5-D9, 1:120 citrate antibody retrieval). In brief, deparaffinized, and rehydrated slides underwent antigen retrieval in 0.01 M citrate buffer at ph 6.0 and were then placed in a 770-W microwave oven for 14 minutes before staining for S-100, EMA, and claudin-1. Proteinase K digestion for 4 minutes was performed for NFP. The slides were stained on the Dako Autostainer (Dako Corporation, Carpinteria, CA) using the EnVision+ (Dako) staining reagents. Positive staining was defined as dark brown cytoplasmic (S-100, NFP, and EMA), nuclear (S-100), and membranous (claudin-1 and EMA) staining patterns. Results The retrospective search identified 44 appendectomies with appendiceal pathologic findings and 1 colectomy with incidental appendiceal lesion. The appendices were from 26 women and 19 men. The mean age of the patients was 50 (range = 1 month to 87) years. The appendiceal diagnosis included 24 cases of diverticular disease with (21) or without (3) superimposed acute inflammation, 17 serrated polyps with (8) or without (9) concurrent inflammation, and 4 mucosal hyperplasia (4). Of 4 mucosal hyperplasia, 2 showed concurrent low-grade dysplasia involving the surface epithelium, characterized by nuclear hyperchromasia and enlargement with increased mitotic figures in the absence of significant inflammation. Two diverticular cases harbored goblet cell carcinoid and conventional carcinoid, respectively, and 1 case was associated with a serrated polyp in the proximal appendix. The control group of normal ( incidental ) appendices without pathologic appendiceal lesions included 27 women and 18 men, with a mean age of 52 (range = 1 month to 89) years. Of the normal appendices, 34 were obtained from colectomies, 10 from oophorectomies, and 1 from an appendectomy. H&E Review In the diverticular disease group, mucosal spindle cell proliferation was identified in 10 cases (42%) from 6 women and 4 men, with a mean age of 44 (range = 22-63) years (Table 2). The distribution of the spindle cell proliferation was diffuse in 3 cases, patchy in 2, and focal in 5 cases. The proliferation was identified in the nonherniated portion of the mucosa. In 4 cases, the proliferation was identified in the diverticular mucosa as well. The spindle cells demonstrated wavy, eosinophilic cytoplasm and bland nuclei. The border between the spindle cell proliferation and the lamina propria was indistinct and irregular. No nuclear pleomorphism, atypia, or mitosis was present. No granulomas, necrosis, or ganglion cells were identified (Figure 1). The adjacent lymphoid follicles were atrophic. In general, the proliferation was conspicuous in the deep mucosa along the muscularis mucosae layer, occasionally extending into the middle or apical portion of the lamina propria, separating apart the adjacent crypts. In 3 cases, the spindle cells were focally clustered underneath the surface epithelial layer associated with a paucity of the underlying crypts. One case (case 10 in Table 2) showed multifocal dense spindle cell proliferations that expanded the lamina propria and separated the adjacent crypts apart (Figure 2A). Also, 6 cases were associated with hyperplastic changes of the overlying surface epithelium (Table 2, Figure 2B). Among the 14 cases of diverticular disease without mucosal spindle cell proliferation, 2 were associated with goblet cell carcinoid and conventional carcinoid, respectively. The remaining 12 cases were not associated with other noninflammatory pathologic conditions. Among the 4 cases of mucosal hyperplasia, mucosal spindle cell proliferation was found in 2 cases with concurrent surface low-grade dysplasia. One of these lesions (case 11 in Table 2) was originally reported as a serrated adenoma. Although there was mild crypt architectural distortion under the undulating mucin-rich surface epithelium, no diagnostic features of a serrated polyp were noted on review. No mucosal spindle cell proliferation was seen in the remaining 2 cases of mucosal hyperplasia without surface dysplasia, and the 17 serrated polyps. The additional case of serrated polyp (case 9 in Table 2, included in the diverticular group) was found in a diverticular case with mucosal spindle cell proliferation. However, the spindle cell proliferation was noted away from the polyp, not within the polyp. In the control group, 9 of 45 (20%) normal appendices showed mucosal spindle cell proliferation, in 3 women and 6 men, whose mean age was 61 (range = 33-89) years (Table 2). There was a statistically significant difference in the incidence of intramucosal spindle cell proliferation in the 3 cohorts: diverticula (42%), serrated polyp (0%), and normal (20%) P =.006, Fisher s exact probability test. Immunoperoxidase Stain The mucosal spindle cells were strongly immunoreactive for anti-s-100 antibody in the nuclei and perinuclear cytoplasm (Figure 2C). In addition, the S-100 immunostain highlighted numerous interconnecting networks of short neural fibers in the lamina propria, which were not recognizable as distinct spindle cell proliferation in the corresponding H&E sections. Two cases showed rare

4 606 International Journal of Surgical Pathology 21(6) Table 2. Cases With Mucosal Schwann Cell Proliferation. a Age G Association Distribution Location Density EH AD 1 30 F Diverticulum Focal Apical Dense Y Y 2 58 M Diverticulum Focal Full thickness Loose N Y 3 22 M Diverticulum Patchy Full thickness Loose Y Y 4 36 F Diverticulum Focal Base and mid Dense Y N 5 58 F Diverticulum Focal Base Loose N Y 6 34 F Diverticulum Diffuse Base and mid Dense N Y 7 63 M Diverticulum Diffuse Base and mid Loose N Y 8 39 F Diverticulum Focal Base and mid Loose Y Y 9 55 F Diverticulum b Patchy Full thickness Loose Y Y M Diverticulum Diffuse Full thickness Loose to dense Y Y F Surface LGD Diffuse Full thickness Loose Y Y M Surface LGD Patchy Full thickness Loose N Y M Abdominal pain Diffuse Full thickness Dense Y Y M Colon adenoma Patchy Base and mid Loose Y N M Colon cancer Patchy Full thickness Dense N N M Intussusception Patchy Full thickness Loose N N M Colon cancer Focal Base and mid Dense N N F Colon obstruction Patchy Base and mid Dense Y Y M Infectious colitis Diffuse Base and mid Loose Y Y F Ovarian tumor Patchy Full thickness Dense Y Y F Colon cancer Patchy Base Loose Y Y Abbreviations: G, gender; EH, epithelial hyperplastic change; AD, crypt architectural distortion; LGD, low-grade dysplasia; F, female; M, male; Y, yes; N, no. a Cases 1-10, diverticulum group; cases 11 and 12, mucosal hyperplasia; cases 13-21, normal control group. b There was a concurrent serrated polyp. The Schwann cell proliferation was identified away from the polyp. NFP-positive axon fibers mixed in the mucosal spindle cell proliferation. EMA and claudin-1 immunoperoxidase stain were negative in the mucosal spindle cells studied. Discussion The vast majority of cases of appendiceal diverticular disease are acquired, and congenital forms of the disease are extremely rare. 2 Recent studies demonstrating an association between appendiceal diverticula and a variety of appendiceal neoplasms has brought renewed interest to this benign entity. 2-4,18 However, the gross and microscopic identification of an appendiceal diverticulum can be challenging, and when not recognized, the further sampling of the specimen for a possible coexisting neoplasm will not typically be performed. In contrast, the underlying mucosal architectural distortion associated with a diverticulum of the appendix may be misinterpreted as a mucinous neoplasm and cause overtreatment. Also, the coexistence of appendiceal low-grade mucinous neoplasms and diverticula is common, which may complicate the interpretation. 2 Thus, accurate gross and microscopic identification of appendiceal diverticular disease can be of critical importance in deciding the management of patients with these lesions. Our study showed that 42% of the appendiceal diverticular disease cases harbored mucosal Schwann cell proliferation. The Schwann cell proliferations were identified within the nonherniated appendiceal mucosa but not necessarily within the diverticula. Thus, the proliferations appear to represent a secondary response to the same chronic insult that is accountable for the diverticula formation, rather than being a direct cause of diverticula. The coexisting acute inflammation seems independent of the mucosal Schwann cell proliferation, given that 8 of 17 appendices with serrated polyps also showed acute inflammation without mucosal Schwann cell proliferation. It is noteworthy that 2 appendices with mucosal hyperplasia and concomitant mucosal Schwann cell proliferation also harbored surface low-grade dysplasia. Hsu et al 3 distinguished appendiceal mucosal hyperplasia with reactive atypia from low-grade mucinous neoplasms by the degree of architectural complexity. In mucosal hyperplasia, the architectural change is prominent in the upper half of the mucosa associated with crypt disarray, whereas the mucosa of a low-grade mucinous neoplasm usually demonstrates a distinct villiform change and back-to-back crypts. The architectural changes seen in our 2 cases of surface low-grade dysplasia associated with mucosal Schwann cell proliferation were subtle and superficial,

5 Stockl et al 607 Figure 1. A. Proliferation of bland spindle cells in the base and midportion of the lamina propria resulting in separation of the adjacent appendiceal crypts. B. Higher magnification view shows the absence of nuclear atypia, pleomorphism, mitosis, and necrosis: A, hematoxylin and eosin (H&E), 100; B, H&E, 200. similar to that encountered in nondysplastic mucosal hyperplasia. Potentially, these 2 cases may represent the very early stages of appendiceal low-grade mucinous neoplasms, in which the mucosal architectural complexity is not yet fully developed. Regardless, appendiceal mucosal Schwann cell proliferation seen in mucosal hyperplasia may coexist with surface low-grade dysplasia, a finding that clearly requires further validation. Lamps et al 2 showed that 42% of the appendiceal lowgrade mucinous neoplasms had coexisting diverticula. They postulated 2 possible routes of pathogenesis: (1) involvement of preexisting diverticula by the low-grade mucinous neoplasm or (2) increased luminal pressure by the mucin from the neoplasm, followed by diverticula formation. If our 2 cases of Schwann cell proliferation and surface low-grade dysplasia indeed represented an early stage of low-grade appendiceal mucinous neoplasm, our observation seems supportive of the second hypothesis of Figure 2. A. Lower magnification view of a diverticular disease showing multifocal spindle cell proliferations (arrows) in the lamina propria associated with distorted crypts. No lymphoid follicles are seen. B. Adjacent mucosa with epithelial hyperplastic change. C. The spindle cells are S-100 immunoreactive. A, hematoxylin and eosin (H&E), 25; B, H&E, 100; C, S-100, 100. Lamps et al. Preexisting mucosal hyperplasia with concurrent surface dysplasia may be complicated by luminal mucin accumulation followed by diverticula formation, given the presence of mucosal Schwann cell proliferation in both. The significance of this observation needs to be further studied. It is also interesting that none of the 17 serrated polyps showed mucosal Schwann cell proliferation within the polyp. Likewise, the additional serrated polyp with concurrent diverticulum (case 9 in Table 2) did not show mucosal Schwann cell proliferation within the stroma of the polyp; the proliferation was away from the polyp. Thus, despite the common finding of mucosal Schwann cells in the appendix, stromal Schwann cell proliferationdriven polyp appears to be rare in the appendix. Moreover, the stromal cells of the appendiceal polyps were negative for perineurial markers such as claudin-1 and EMA. Hence, perineurioma and fibroblastic polyp are uncommon in the appendix. 19 Two-thirds of the appendices with mucosal Schwann cell proliferation showed epithelial hyperplasia (Table 2).

6 608 International Journal of Surgical Pathology 21(6) It is conceivable that some cases of epithelial or mucosal hyperplasia in the setting of mucosal Schwann cell proliferation may be interpreted as a hyperplastic polyp or serrated polyp, as demonstrated in the current study. Awareness of the association of mucosal Schwann cell proliferation with epithelial hyperplasia and the lack of association between the proliferation and true serrated polyps may help avoid this misinterpretation. The fact that 20% of the normal adult appendices and 42% of the diverticular disease showed mucosal Schwann cell proliferation, whereas none of the true epithelial proliferation-driven polyps did adds credence to the benign and acquired nature of the appendiceal mucosal Schwann cell proliferation. 5,8 However, the association of the proliferation with diverticular disease and surface low-grade dysplasia signify the importance of recognizing the phenomenon. Although the appendiceal mucosal Schwann cell proliferation is neither a sensitive nor a specific histologic marker of appendiceal diverticular disease, it may justify further examination of the remaining appendix specimen. Additional sampling may reveal diverticula and/or neoplasms associated with diverticula, or epithelial dysplasia. Several studies have reported an association of appendiceal diverticular disease with a regional concomitant neoplasm in up to 48% of the cases. 4,18 In the current study, there were only 2 cases of appendiceal neoplasm goblet cell carcinoid and conventional carcinoid and a serrated polyp among the 24 diverticula cases. The low incidence of coexisting appendiceal neoplasm in our study in comparison with other published reports may be a result of selection bias, especially the exclusion of low-grade mucinous neoplasms. Moreover, in the cases of obvious neoplasm, additional benign lesions such as diverticula may be ignored or underrecognized and may not be reported. Because of the retrospective nature of this study, we were unable to submit additional sections of the appendices with mucosal Schwann cell proliferation. Therefore, although our observation raises a possibility that mucosal Schwann cell proliferation, a putative histologic marker of appendiceal diverticula, may be associated with occult appendiceal neoplasms, the hypothesis needs validation with further prospective studies. In summary, this study found that 42% of the appendiceal diverticula cases and 20% of normal appendices showed mucosal Schwann cell (neural) proliferation. The proliferation may be a putative histologic marker of diverticula, their associated neoplasms, and epithelial dysplasia. We conclude that the awareness of this association in an appendix with mucosal Schwann cell proliferation that has undergone a routine pathologic examination justifies the need for additional sampling of the specimen to exclude significant appendiceal disorders, which may subsequently require changes in patient management. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. References 1. Abdullgaffar B. Diverticulosis and diverticulitis of the appendix. Int J Surg Pathol. 2009;17: Lamps LW, Gray GF, Dilday BR, Washington MK. The coexistence of low-grade mucinous neoplasms of the appendix and appendiceal diverticula: a possible role in the pathogenesis of pseudomyxoma peritonei. Mod Pathol. 2000;13: Hsu M, Young RH, Misdraji J. 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7 Stockl et al Franke C, Gerharz CD, Böhner H, et al. Neurogenic appendicopathy: a clinical disease entity? Int J Colorectal Dis. 2002;17: Kallenbach K, Hijorth SV, Engel U, Schlesinger NH, Holck S. Significance of acquired diverticular disease of the vermiform appendix: a marker of regional neoplasms? J Clin Pathol. 2012;65: Groisman GM, Polak-Charson S. Fibroblastic polyp of the colon and colonic perineurioma: 2 names for a single entity? Am J Surg Pathol. 2008;32:

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