Laboratory Animals–Original Article

Immunohistochemical Characterization of Large Intestinal Adenocarcinoma in the Rhesus Macaque (Macaca mulatta)

Veterinary Pathology 2015, Vol. 52(4) 732-740 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985814556188 vet.sagepub.com

C. E. Harbison1, F. Taheri1, H. Knight2, and A. D. Miller3

Abstract In rhesus macaques, adenocarcinomas of either the ileocecal junction or colon are common spontaneous tumors in aging populations. The macaque tumors have similar gross and histologic characteristics compared with their human counterpart, but little is known regarding the immunohistochemical expression of proteins that are commonly implicated in the pathogenesis of these tumors in humans. We performed a retrospective review of 22 cases of large intestinal carcinoma in the rhesus macaque and evaluated the expression pattern of a panel of potentially prognostically significant proteins identified from human studies. Histologic characteristics of the tumors included abundant mucin deposition, transmural spread, and lymphatic invasion. All rhesus adenocarcinomas displayed altered expression of 1 or more of CD10, b-catenin, sirtuin 1, cytokeratin 17, and p53 compared with age-matched controls. Zymographic analysis of active matrix metalloproteinases 2 and 9 in the serum from 5 animals failed to reveal statistically significant differences between adenocarcinoma cases and controls. Based on the data presented herein, large intestinal carcinomas in the macaque share many histomorphologic and immunohistochemical similarities to large intestinal tumors in humans. Further validation of this animal model is considered important for the development of novel therapeutics and a better understanding of the pathogenesis. Keywords macaque, immunohistochemistry, cd10, intestine, sirtuin 1, b-catenin, mucinous adenocarcinoma, colon, cecum

In humans, large intestinal adenocarcinoma (liAC) is the most common tumor of the intestine and is the third leading cause of cancer death in the United States.39 Risk factors for liACs include age (>50 years), sex (men slightly overrepresented), smoking, obesity, dietary factors such as low fiber and high red meat consumption, and a history of ulcerative colitis or inherited polypoid syndromes.34 Two-thirds of these tumors are located in the rectum or distal (sigmoid) colon.39 Human liACs tend to develop slowly over a period of years and, in the inherited forms, commonly progress from benign polyps and/or adenomas to malignant tumors that invade transmurally through the colon wall. Metastases are common via lymphatics and/or blood vessels to distant sites, including draining lymph nodes, liver, and abdominal serosa (carcinomatosis).6 Many genetic mutations and epigenetic modifications leading to tumor progression have been identified in genes such as KRAS, adenomatous polyposis coli (APC), CTNNB1, and p53.35 In the rhesus macaque, large intestinal carcinomas are similarly common, occurring with roughly equal incidence at the ileocecal junction and proximal colon.2 Large intestinal carcinomas tumors account for up to 50% of tumors identified in animals older than 20 years (rhesus macaque life span approximately 25–30 years).38,42 As opposed to the human condition, progression from polyps and/or adenomas to carcinomas has not been

recognized, and tumors of the rectum are uncommon in the macaque.36,42,43 In addition, there appear to be differences in the genetic basis of tumorigenesis, as examination of KRAS failed to find mutations in any of 10 animals examined in one study, compared with a 40% mutation rate in humans.43 Despite these differences, the course of disease proceeds similarly to that of humans, with a similar gross and histologic appearance, including frequent deposition of mucin, slow transmural invasion, carcinomatosis, and/or metastasis to regional lymph nodes and distant organs via both lymphatics and blood vessels.36 In addition, the

1 New England Primate Research Center, Division of Comparative Pathology, Harvard Medical School, Southborough, MA, USA 2 Abbvie Bioresearch Center, Worcester, MA, USA 3 Cornell University College of Veterinary Medicine, Department of Biomedical Sciences, Section of Anatomic Pathology, Ithaca, NY, USA

Supplemental material for this article is available on the Veterinary Pathology website at http://vet.sagepub.com/supplemental. Corresponding Author: A. D. Miller, Cornell University College of Veterinary Medicine, Department of Biomedical Sciences, Section of Anatomic Pathology, T5-006A Veterinary Research Tower, Ithaca, NY 14853, USA. Email: [email protected]

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clinical signs associated with intestinal adenocarcinomas in both species are similar and include weight loss, changes in appetite or bowel movements, and the intermittent presence of occult blood in the stool.36 A wide variety of prognostic features have been evaluated in human liACs, including histologic characteristics, altered protein expression patterns within tumors, and changes in the levels of proteins within serum. Recently, several studies used techniques such as genome-wide microarray and proteomics to evaluate global gene expression changes in liACs to identify diagnostically and prognostically important markers of disease.6,9 Several of the more important genes/proteins that have altered expression in human liACs include cytokeratin 17 (CK17), sirtuin1 (SIRT1), b-catenin, CD10, matrix metalloproteinases (MMPs), and p53.15,19,24,25,41,45 CK17 is a marker of basal epithelia and defines progression in several human cancers, including squamous cell, mammary, ovarian, and gastric carcinomas, where it is frequently associated with poorer prognosis.18,44 SIRT1 has been implicated as both an oncogene and a tumor suppressor gene in various cancers, including human liAC through multiple interactions with cellular pathways such as Wnt signaling, p53, and DNA damage repair, and thus its role in cancer development and progression remains unclear.10 Increased expression of CD10, a zinc metalloendopeptidase, has been correlated with disease progression and invasion in humans, with staining found in only 25% of low-grade tumors but in 60% of high-grade liACs.20 b-Catenin is a tight junction protein, and its altered expression is implicated in various neoplasms via aberrant Wnt signaling.8,26 In human liACs, loss of membranous b-catenin is most frequently observed at the invasive front of tumors and is correlated with a decreased diseasefree survival interval following surgical removal.5,24 p53 is the most commonly mutated tumor suppressor gene in a wide variety of neoplasms, including colon cancer, and plays a role in cell cycle arrest and apoptosis.16 Inactivating mutations lead to increased expression levels in tumor cells, and these have been observed in about 50% of human liACs, with only inconsistent links to poor patient outcome.4,17,29,40 To our knowledge, these markers have not been evaluated in liACs in the rhesus macaque, and therefore the role that altered expression may play in tumorigenesis is unknown. The current study summarizes the clinical and histopathologic features of 22 cases of large intestinal carcinoma and examines the immunohistochemical expression patterns of CK17, SIRT1, CD10, b-catenin, and p53 to determine if similar alterations exist in large intestinal carcinomas in the macaque. Serum samples were also evaluated for MMP-2 and MMP-9 enzyme activity to evaluate their utility as a biomarker for animals that are presumed to have large intestinal carcinoma.

Materials and Methods The database of necropsy and biopsy case records at the New England Primate Research Center (NEPRC) was searched from 1997 to 2012 and reviewed for cases of large intestinal adenocarcinoma and/or carcinomatosis of intestinal origin. All

animals included in this study were cared for in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th edition, 2011) and the standards of the Harvard Medical School Standing Committee on Animals and the Association for the Assessment and Accreditation of Laboratory Animal Care. All animals were necropsied within 24 hours of death, and representative sections of intestinal lesions or normal colon were collected, fixed in 10% neutral buffered formalin (NBF), and paraffin embedded according to standard procedures. Then, 5-mm histologic sections were routinely processed and stained with hematoxylin and eosin (HE). One to 3 sections of large intestine, 1 section of mesenteric lymph node, and 1 to 2 sections of other organs were evaluated, targeting grossly evident lesions when present. Cases of liAC were classified as mucinous if the tumors contained 1 or more areas of mucinous differentiation and were subdivided according to the current American Joint Committee on Cancer (AJCC) staging system, with the salient features summarized as follows: stage 0, no invasion into the muscularis mucosae (carcinoma in situ); stage I, invasion into the bowel through the muscularis mucosae into the submucosa; stage II, invasion into or through the smooth muscle layers without spread to distant sites; stage III, invasion through the muscle layers to the serosal surface, pericolonic tissues, and/or nearby lymph nodes; and stage IV, solid organ metastases including to the lung, liver, and/or spleen, with or without carcinomatosis or lymph node involvement.1 To characterize the cases of large intestinal adenocarcinoma using immunohistochemical methods, we used antibodies against the following proteins: cytokeratin 17 (CK17), sirtuin 1 (SIRT1), b-catenin, CD10, and p53. Briefly, all staining protocols used an antigen retrieval protocol of microwaving in sodium citrate buffer for 20 minutes followed by 20 minutes of cooling at room temperature (RT). The source, dilution, and incubation time of the primary antibodies used in these studies are detailed in Supplemental Table S1. Secondary antibodies were biotinylated horse anti–mouse (Vector Laboratories, Burlingame, CA) or biotinylated goat anti–rabbit (Vector Laboratories) antibodies as appropriate, diluted 1:200, and incubated for 30 minutes at room temperature. Tertiary reagent in each case was Vectastain ABC Elite reagent incubated for 30 minutes at RT (Vector Laboratories). All slides were developed with DAB chromogen (Dako, Carpinteria, CA) and counterstained with Mayer’s hematoxylin. Negative controls consisted of unaffected, adjacent sections of large intestine stained with irrelevant species-, isotype-, and concentration-matched antibodies. To assess staining intensity for the above noted antibodies, the following scoring system was developed as compared with control sections of adjacent large intestine: 0, no staining; 1, faint staining of individual cells; 2, staining of scattered cells or aggregates of cells; 3, increased regional or diffuse staining or comparable staining to the control sections (for b-catenin and SIRT1); and 4, increased staining intensity compared with controls (applicable only to SIRT1). Statistically significant differences between staining groups were determined by the Fisher exact test. A Wilcoxon rank-sum test was done to

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determine if there were expression differences between those with and without metastasis.

Serology and Zymography Serum levels of enzymatically active MMP-2 and MMP-9 were determined for cases in which serum was available (5/22) and age-matched controls. Serum samples were collected and centrifuged to remove debris and dead cells. Serum was diluted with Tris-Glycine SDS Running Buffer (Invitrogen/Life Technologies, Grand Island, NY) and mixed with an equal volume of 2 Novex tris-glycine SDS sample buffer (Invitrogen) followed by separation on a 10% zymogram (gelatin) gel from Invitrogen for 4 hours. The gel was then incubated for 1 hour at RT in zymogram renaturing buffer (Invitrogen), followed by a 30-minute equilibration at RT and an overnight incubation at 37 C with gentle agitation in zymogram developing buffer (Invitrogen) before being stained with Simply Blue SafeStain (Invitrogen) for 30 minutes and washed with distilled water to visualize the bands and determine the enzyme activity of MMP-2 and MMP-9 species. The gels were examined on the VersaDoc Imaging System (Bio-Rad, Hercules, CA), and the intensity of the corresponding bands was determined in comparison to that of a recombinant human proMMP-9 dimer standard sample (EMD Millipore, Waltham, MA) loaded at 2.5 mL (mixed with equal volume of the 2 sample loading buffer) on each gel as a reference.

Results Clinical, Gross, and Histopathology Findings The clinical features of 22 cases of spontaneous liAC are summarized in Supplemental Table S2. Of the 22 animals, 14 were on experimental protocols for neurophysiology studies; however none involved experiments that would have been expected to directly affect the development of large intestinal carcinomas. One was inoculated with simian immunodeficiency virus (SIVmac239) with progression to simian acquired immunodeficiency syndrome (SAIDS), and 3 of 22 additional animals were vaccinated with attenuated SIV strains. All masses were identified at necropsy except case No. 7, which was an excisional biopsy, and at necropsy 5 years, later no recurrent or metastatic liAC was identified from this animal. Fourteen of 21 (67%) of the remaining animals either died spontaneously or were euthanized for clinical signs related to the primary tumor, including a palpable abdominal mass, wasting, diarrhea, or signs of intestinal obstruction and bloating. Overall, cases were identified in slightly more females than in males and primarily in aged animals, with 19 of 22 (86%) of animals older than 20 years. The animals’ weights were widely variable, with a range of 4.8 to 16 kg (median, 8.9 kg). Ten tumors were located at the ileocecocolic junction, and 10 were within the large intestine (the location along the length of the colon was generally not further specified). No tumors were identified arising from the rectum. In 2 cases, neoplastic

infiltrates consistent with a large intestinal carcinoma were identified only within the serosa of the large intestine or mesentery (carcinomatosis), and a primary mass was not observed at necropsy. Case Nos. 4 and 10 had concurrent small intestinal carcinomas that were considered distinct neoplasms due to inconsistent histologic features between the 2 tumors. Amyloidosis was the most common spontaneous concurrent disease, present in 1 or more of the spleen, adrenal, stomach, small intestine, colon, kidney, lymph node, or pancreas in a total of 17 of 21 cases. Other common findings included myocardial fibrosis (8/21), chronic lung mite infestation (5/21), chronic interstitial nephritis (5/21), and uterine adenomyosis (2/13). Concurrent neoplasms in addition to the aforementioned small intestinal carcinomas included single cases of biliary adenoma, ovarian adenoma, pheochromocytoma, lipoma, gastrointestinal stromal tumor, uterine leiomyoma, and 2 cases of islet cell adenoma. Generally, neoplastic epithelial cells formed packets, cords, and acini that invaded widely throughout the intestinal muscle layers (Fig. 1). Neoplastic cells had moderate amounts of pale basophilic granular cytoplasm with often basally oriented nuclei that had finely stippled chromatin and 1 to 3 distinct nucleoli. Neoplastic cells were often embedded in a dense scirrhous response (desmoplasia), and there were variable multifocal areas of necrosis and mucosal ulceration with replacement by neutrophils, hemorrhage, and fibrin. Tumors were variably infiltrated by small to moderate numbers of neutrophils, lymphocytes, macrophages, and eosinophils, whereas the lamina propria of the normal colon contained only small numbers of lymphocytes and plasma cells with rare eosinophils. Tumors were histologically staged according to the American Joint Committee on Cancer’s 4-stage classification system.1 Twenty of the 22 tumors had invaded at least completely through the bowel wall at necropsy, with 11 at stage II indicating transmural invasion, 5 at stage III with 2 cases of metastasis to the lymph nodes and 3 with carcinomatosis but no solid organ metastasis (Fig. 2), and 4 at stage IV with distant metastasis to the lung, liver, and/or spleen. Three of these had concurrent carcinomatosis for a total carcinomatosis prevalence of 27%. The overall prevalence of lymph node and/or solid organ metastasis was 6 of 22 (27%). Vascular and/or lymphatic invasion was present in 41% of tumors, including all 6 cases with distant metastasis or carcinomatosis and 3 additional cases in which further tumor spread was not identified (Fig. 3). Of note, none of the animals that were euthanized for study purposes and were clinically healthy were either stage III or IV. Ten tumors (45%) were classified as the mucinous histologic subtype (Fig. 4). No animals with tumors of this subtype had identifiable lymph node or distant metastasis to solid organs, a significant difference from nonmucinous tumors (P < .05). Only 2 of 10 mucinous tumors had vascular invasion vs 7 of 12 nonmucinous liACs, although this difference was not statistically significant due to low number of cases (P ¼ .09). Two cases with carcinomatosis were of the mucinous variant, which was equivalent to the occurrence of carcinomatosis in nonmucinous tumors.

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Figures 1–4. Large intestine; rhesus macaque. Hematoxylin and eosin (HE). Figure 1. Ileocecal adenocarcinoma. Invasive clusters of neoplastic epithelial cells invade into the colonic wall. Figure 2. Ileocecal adenocarcinoma. Transmural invasion by epithelial cells with serosal invasion. Figure 3. Ileocecal mucinous adenocarcinoma. Vascular and lymphatic invasion by rafts of neoplastic epithelial cells (asterisk indicates vessel lumen). Figure 4. Cecocolic mucinous adenocarcinoma. The mucinous histologic subtype is characterized by large lakes of mucin either surrounded by variably attenuated neoplastic epithelial cells or dissecting between muscular layers (asterisk).

Immunohistochemical Findings Immunohistochemistry findings for cytokeratin 17 (CK17), sirtuin 1 (SIRT1), b-catenin (b-cat), CD10, and p53 expression in adenocarcinomas and normal large intestine are presented in Table 1. Histologically normal colon and cecum, as well as normal areas of colon included in tumor sections, were negative for CK17 (Fig. 5). Within liAC cases, cytoplasmic CK17 immunoreactivity was present in the neoplastic cells of 9 of 22 cases, including the 2 youngest animals. CK17 immunoreactivity was scattered in individual or contiguous areas of cells within hyperplastic tortuous crypts, areas of infiltration (Fig. 6), and, rarely, the attenuated cells lining lakes of mucin (Fig. 7). In normal tissues, SIRT1 immunoreactivity was detected within the nuclei of scattered cells in the crypt epithelium as well as occasional inflammatory cells in the lamina propria

(Fig. 8). SIRT1 immunoreactivity was variable across the lesions within the case series. In 4 of 21 cases, the intensity of nuclear staining was focally to multifocally increased compared with control sections in both mucinous and nonmucinous areas (Fig. 9). Interestingly, 3 of these cases were identified incidentally at necropsy, and the remaining clinically apparent case (No. 10) had small areas of increased immunoreactivity with concurrent decreased immunoreactivity in other areas of the neoplasm. Eleven of the remaining 17 cases had slight to marked reduction in the overall intensity of SIRT1 immunoreactivity, also with some variability across the tumor with large areas of tumors designated staining intensity 1 with nearly complete loss of expression (Fig. 10). Normal large intestinal epithelium was uniformly negative for CD10 (Fig. 11). Immunoreactivity for CD10 was frequently detected in neoplastic enterocytes in 12 of 22 liAC cases, either exclusively on the apical membrane of neoplastic epithelial

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Table 1. Immunohistochemical Staining Localization and Intensity Scoring of Large Intestinal Adenocarcinomas in 22 Rhesus Macaques. Case No.

CK17

SIRT1

b-Catenin

CD10

p53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Total altered C23–C27a

0 1 2 0 0 3 0 0 2 0 0 1 0 2 3 3 0 0 0 0 0 3 9/22 0

1 2–3 3 4 4 3 3 1 4 2–4 2 1 1 2–3 3 3 1 2 2 1 3 3 15/22 3

2 CP 1 3 3 3 1 2 1 2 1 CP 1 1 1 3 3 3 n/d 3 1 CP 1 CP 1 CP 3 13/21 3

2 CP 1 3 0 1 CP 2 CP 0 0 0 0 3 0 0 3 0 1 CP 2 CP 1 0 2 CP 0 2 12/22 0

0 3 3 2 2 2 3 1 3 1 3 3 0 3 2 1 1 2 0 2 n/d 2 18/21 0–1

The total number of cases with altered staining compared with controls is indicated. CP, aberrant cytoplasmic staining; n/d, staining not performed due to tissue limitations. a The 5 control (case Nos. C23, C24, C25, C26, and C27) are grouped together as each had similar staining properties.

cells (Fig. 12) or in a granular pattern within the apical cytoplasm in 6 cases (Fig. 13) and rarely around mucinous areas. In control sections and nontumorous regions, b-catenin had a membranous immunoreactivity pattern in epithelial cells (Fig. 14). The intensity of b-catenin membrane immunoreactivity was reduced in most tumors examined (14/21) (Fig. 15) with concurrent accumulation in the cytoplasm and/or indistinctly in the nucleus in 5 cases (Fig. 16). Staining of controls for p53 was either negative or had rare scattered cells within the crypts with faint nuclear immunoreactivity (Fig. 17). Enhanced nuclear p53 immunoreactivity was detected in almost all of the tumors (18/21) with variable intensity. In 4 cases, immunoreactivity was positive in scattered neoplastic cells only (Fig. 18), whereas in the remaining positive tumors, there was a locally extensive to regional, multifocal immunoreactivity of adjacent cells forming acini (Fig. 19). For each immunohistochemical marker, no association of any staining distribution or intensity was identified with mucinous phenotype, tumor stage/presence of distant metastasis, or location (ileocecal junction vs colon). In total, compared with control cases, 1 case had alterations in all 5 markers examined, 6 cases had alterations in 4 of the markers, 9 cases in 3 markers, 5 cases in 2 markers, and 1 case (where the primary tumor was not identified) had only 1 marker altered.

Serum Levels of Active Matrix Metalloproteinases The level of enzymatically active MMP-2 and MMP-9 within the serum was determined in 5 cases and 4 age-matched controls. No significant differences between liAC cases and controls were observed for either enzyme (P > .05) (data not shown). Interestingly, in the 1 case of excisional biopsy (No. 7), the prebiopsy sample was higher than the terminal serum sample for both enzymes, with a greater reduction in MMP-9 vs MMP-2 (Suppl. Fig. S1).

Discussion In humans, adenocarcinomas of the large intestine are one of the most common tumors and are associated with a variety of etiologies, including dietary and genetic factors. Rhesus macaques are used as the preeminent model for a variety of human conditions, and although large intestinal carcinomas are common in rhesus macaques, in-depth comparison of histologic and immunohistochemical expression patterns has not been performed. These 22 cases had evidence of dysregulated signaling pathways commonly implicated in the development of intestinal neoplasia, such as Wnt and p53, and subsets of cases shared altered expression of proteins identified as prognostically important in humans. The most common pattern was concurrent increased p53 staining, decreased SIRT1 staining, and decreased b-catenin, present in 10 of 22 cases. There was, however, marked variability between cases in the distribution and intensity for each of the tested proteins. Similar to previous reports, the liACs in aged rhesus macaques reported herein arose most commonly at the ileocecal junction and along the large intestine. No tumors were found arising from the rectum. No polyps were identified, nor were polypoid areas adjacent to the neoplasms found suggestive of a progressive lesion as has been reported in humans.34 In addition, unlike cases of liAC reported in cotton top tamarins, no evidence of concurrent colitis was identified and therefore no association with inflammatory bowel disease can be concluded.21 Although rarely performed on animals in this study, the index of suspicion for this tumor was high if fecal occult blood tests were positive, especially when combined with a history of weight loss (data not shown). In humans, detection of tumors in the early stages of disease via regular screening by colonoscopy or fecal occult blood tests allows for surgical resection that is overwhelmingly curative. To that point, the single case of carcinoma in situ reported herein that was treated by excisional biopsy did not recur. Reported histologic features significantly related to prognosis in humans include the depth of neoplastic infiltration into the colonic wall, lymphatic or vascular invasion by neoplastic cells, and tumor location, with lesions in the lower third of the rectum having a worse prognosis than those at other sites.31 Overall, liACs in rhesus macaques are highly locally invasive and readily metastasize. Nearly all of the neoplasms in the current study set were of AJCC stage II or greater, indicating at least transmural invasion of neoplastic cells. This finding is consistent with the

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Figures 5–10. Large intestine; rhesus macaque. Immunohistochemistry. Figure 5. Normal colon. A representative section of control large intestine is negative for cytokeratin 17. Figure 6. Colonic adenocarcinoma. Variably intense intracytoplasmic cytokeratin 17 immunoreactivity is present in the acini of neoplastic cells. Figure 7. Mucinous adenocarcinoma. Cytokeratin 17 immunoreactivity is rarely present in the neoplastic cells lining the mucin deposits (inset: higher magnification). Figure 8. Normal colon. Sirtuin 1 immunoreactivity is present in crypt epithelium and inflammatory cells in the lamina propria. Figure 9. Colonic mucinous adenocarcinoma. Markedly increased nuclear immunoreactivity for sirtuin 1 in neoplastic epithelial cells. Figure 10. Colonic mucinous adenocarcinoma. Decreased and varied nuclear staining intensity for sirtuin 1 in neoplastic epithelial cells.

development of serious clinical signs in most animals with these tumors, necessitating euthanasia or resulting in the spontaneous death of the animal. However, the observation that 5 animals were clinically healthy and had liACs of stage II underscores that these tumors can progress insidiously and may remain undetected for a period of time (up to years in humans) unless active screening programs are in place. The percentage of tumors with metastases was similar to previous reports in macaques, while the occurrence of carcinomatosis was higher at 27%.39 Overall, vascular and/or lymphatic invasion was common, suggesting a high probability of eventual metastasis given the continued survival of the animal. In addition, the evaluation of vascular invasion and grossly inapparent distant metastasis may be underestimated due to sampling limitations. Future longitudinal studies are required to determine the rate of progression and survival curves in rhesus macaques. The mucinous variant of liAC accounts for 10% to 20% of all colorectal cases in humans, is more common in younger patients in the proximal colon, and may be associated with more severe disease.23 This variant is characterized by large lakes of extracellular mucin that is secreted by the neoplastic cells and has been postulated to dissect through the colonic wall, leading to increased invasiveness.11 Mucinous liACs appear to be more common in rhesus macaques than in humans overall, representing a range from *30% to 100%

of liAC cases in several previous studies and in 45% in the current study.36,38,42 No association of histologic subtype with outcome was postulated in previous macaque studies based on small sample sizes, a similar limitation of the current study. However, some evidence presented herein suggests this variant may actually be less invasive, as no mucinous liAC cases had lymph node or distant solid organ metastasis. Genome-wide analyses of human large intestinal adenocarcinomas have revealed several gene and protein alterations of diagnostic and prognostic significance, including CK17, SIRT1, CD10, b-catenin, and p53.9,25 CK17 immunoreactivity was noted in 41% of liAC cases in the current study set, including the 2 youngest animals. It was also commonly observed in those animals that died with gastrointestinal (GI) signs (6/14, 43%) and thus may be an indicator of later stages of disease progression. In 1 study in humans, CK17 reactivity was present in 68% of malignant intestinal neoplasms and trended toward (but did not reach significance for) increased expression both at the invasive front and in higher stage tumors. Thus, this marker warrants additional investigation to establish clearer ties to prognosis.25 SIRT1 expression levels were higher in the single case of carcinoma in situ and in 2 cases in which the tumors were incidental findings at necropsy, as well as in small areas of a primary tumor that also had distant metastasis. In most other

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Figures 11–19. Large intestine; rhesus macaque. Immunohistochemistry. Figure 11. Normal colon. The epithelium of normal colon has no immunoreactivity for CD10. Nonspecific staining is present in the cytoplasm of inflammatory cells within the lamina propria (inset). Figure 12. Cecocolic mucinous adenocarcinoma. A subset of cases was characterized by neoplastic cells with strong apical (inset: higher magnification) immunoreactivity for CD10. Figure 13. Colonic adenocarcinoma. A subset of cases was characterized by cytoplasmic immunoreactivity for CD10 within the neoplastic epithelial cells. Figure 14. Normal colon. There is strong, membranous b-catenin immunoreactivity. Figure 15. Colonic adenocarcinoma. A marked decrease in membranous b-catenin immunoreactivity within neoplastic acini (inset: higher magnification). Figure 16. Colonic mucinous adenocarcinoma. Reorganization of b-catenin immunoreactivity to the cytoplasm (inset: higher magnification). Figure 17. Normal colon. Normal colon has no immunoreactivity for p53 with the exception of rare faint positive cells in one control (inset). Figure 18. Ileocecal mucinous adenocarcinoma. Increased nuclear p53 immunoreactivity is present in neoplastic cells lining mucin deposits (inset: higher magnification). Figure 19. Colonic adenocarcinoma. Increased nuclear p53 immunoreactivity is present in large numbers of acini.

tumors (65% overall), SIRT1 staining intensity was reduced compared with controls. These findings are most consistent with its suggested role as a tumor suppressor gene in human liACs, in which retention of SIRT1 expression was associated with overall increased survival and good prognosis and loss of SIRT1 were associated with increasing grade and metastasis.19,22 Alternatively, SIRT1 expression may play an oncogenic role in the early stages of tumor progression for some subsets of tumors.27,28 Unlike in humans, in whom SIRT1 loss is associated with mucinous subtype, no association of SIRT1 expression was seen with this phenotype in the macaque.19

Increased expression of CD10 has been found in cases of human colorectal adenocarcinomas and has been correlated with the presence of hepatic metastasis.12 CD10 expression was present in most rhesus tumors, including the 2 cases with liver metastasis, but not in the tumors of stage 0 or 1, and thus may also be a marker of more advanced disease. The significance of the different staining patterns (membranous vs cytoplasmic) is unclear. In previous human studies, invasive tumors showed increased expression overall, with well-differentiated epithelial tumors having a membranous staining pattern and poorly differentiated tumors showing redistribution to the cytoplasm.7

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Alterations in b-catenin expression in rhesus liACs are comparable to those previously reported in humans, with both loss of membranous expression and/or redistribution to the interior of the cell in a majority of cases. In human cancers, aberrant Wnt signaling is a frequent early event in the progression to liAC and is most commonly activated by mutations in the upstream regulatory gene complex member adenomatosis polyposis coli (APC).5,14,33,37 To our knowledge, no survey of APC gene mutations has been performed in rhesus, and therefore whether it plays a role in tumorigenesis in the macaque is still unknown. In the current study, increased p53 expression was seen in most tumors (18/21, with staining intensity 2 or 3 in 14 cases), consistent with its well-established role in tumor progression, but was not associated with histologic pattern or other features and is thus a less useful histochemical marker than others used in this study. This is in contrast to studies in humans, in which 50% of tumors overall showed accumulation of p53 with the mucinous variant of tumors expressing less p53 than other histologic patterns.32 Examination of serum markers for tumor detection and prognosis is of increasing interest as an improved noninvasive alternative to traditional screening methods such as fecal occult blood test and colonoscopy.9 Higher levels of MMPs, such as MMP-9, are reported within human colonic adenocarcinomas and in patients’ serum, although direct links to prognosis have not been consistently established.3,30 In the current study, no correlation between serum MMP-2 and MMP-9 levels in animals with and without liACs was observed. Of note, however, the levels of both these markers decreased in the 1 excisional biopsy case after removal of the tumor. While more cases are required to determine the significance of this finding, this initial result parallels that seen in humans where the markers are not always informative from a diagnostic perspective but may be useful for tracking the success of tumor resection and/or recurrence within an individual.13 The current study describes the histologic and immunohistochemical features of 22 cases of ileocecal and colonic adenocarcinoma in the rhesus macaque. Increased monitoring of aging rhesus populations to identify early tumors for longitudinal studies will be essential to determine the importance of mutations of these and other prognostic markers at different stages of neoplastic progression. While not conclusive, preliminary findings from this study suggest that SIRT1 may show enhanced expression in earlier stages of rhesus liACs, while expression of CD10 and CK17 may increase with progressive disease. Furthermore, the establishment of diagnostically relevant serum markers remains a challenge in both nonhuman primate and human liAC cases and should be an area of further study. Acknowledgements We are grateful for the necropsy assistance provided by Elizabeth Curran and Michael O’Connell. We thank Kristin Toohey and Jen Patterson for assistance with images. We greatly appreciate the assistance provided by Dr. Hollis Erb with statistical analysis.

Author Contribution Conception or design: CEH, FT, HK, ADM. Data acquisition, analysis, or interpretation: CEH, FT, HK, ADM. Drafting the manuscript: CEH, FT, HK, ADM. All authors participated in critically revising the manuscript, gave final approval, and agree to be accountable for all aspects of work to ensure integrity and accuracy.

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) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded, in part, by National Institutes of Health grants NEPRC base grant OD0111103 and T32 OD011064.

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Immunohistochemical Characterization of Large Intestinal Adenocarcinoma in the Rhesus Macaque (Macaca mulatta).

In rhesus macaques, adenocarcinomas of either the ileocecal junction or colon are common spontaneous tumors in aging populations. The macaque tumors h...
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