Malignant Neoplasia of the Sex Skin in 2 Chimpanzees (Pan troglodytes)

Abstract

This report describes 2 cases of spontaneous malignant neoplasia within the sex skin of aged female chimpanzees. In both cases, the initial presentation resembled nonhealing traumatic wounds to the sex skin, with different degrees of infection, ulceration, and tissue necrosis. Histopathology of the lesions confirmed the diagnosis of squamous cell carcinoma in one case and of adenocarcinoma with metastasis in the other. Advanced age and previous trauma likely contributed to the development of the neoplasias in both cases; long-term sun exposure may also have contributed to the development of the squamous cell carcinoma. To our knowledge, these 2 cases represent the first reports of sex skin neoplasia in chimpanzees.

The term ‘sexual skin’ was first used in 1891 to describe the cyclically swelling skin on the buttocks and thighs of rhesus macaques.⁶ Since then, prominent sexual swellings have been described in multiple NHP species, including many cercopithecines, some colobines, and chimpanzees.^9-11,30 In chimpanzees, the sex skin is a thin, distensible, hairless, and hypopigmented region of skin that surrounds the vagina and extends dorsally around the anus.^27,34 Sex skin swelling during the menstrual cycle function to increase female attractiveness and stimulate male sexual arousal.^10,30,34 The onset of cyclical sex skin tumescence also is used to detect puberty in chimpanzees, which generally occurs in females between the ages of 8 and 11 y.²¹

In NHP species that exhibit pronounced swellings, such as chimpanzees, there is often extensive variation in swelling size both among females and between the cycles of individual females.¹¹ Swelling increases progressively during the follicular phase of the estrous cycle, as the sex skin becomes smooth, shiny, and turgid and the color changes from dull pink to bright red.^6,34 Histologic examination of sex skin during the follicular phase reveals a thin stratum corneum and dilated dermal blood vessels.⁶ The skin remains at maximal turgescence for 5 to 6 d before the swelling begins to decrease during the luteal phase.³⁴ The bright color rapidly fades, the skin becomes flaccid and wrinkly and attains a scaly appearance due to a thickened stratum corneum.^6,34 There is then a resting phase before menstruation begins again, and during this stage, some females slough areas of epithelium.³² Traditionally, maximal sexual swelling (1 to 6 d prior to detumescence) has been an accepted marker to signal ovulation in chimpanzees.^2,21,30,34 However, this association is not certain, given that ovulation has been documented to occur at other times during the estrous cycle and in view of reports of sexual swelling during infertile times, such as adolescence and pregnancy.^2,9,26,35

Although spontaneous neoplasia was once considered to be uncommon in NHP and especially in chimpanzees, cases are increasingly reported as captive primate colonies age.^3,828,31 A report of all neoplasia documented at 2 major chimpanzee facilities from inception through April 2008 and including both spontaneous and experimental tumors revealed that neoplasia was not uncommon in chimpanzees and that, although most tumors were benign, malignancies did occur.³ The incidence was much higher in female than male chimpanzees due to the high incidence of neoplasia in the female urogenital system, particularly uterine leiomyoma.³ However, in that report of 117 tumors, no sex skin or perineal neoplasms were documented.³ Sporadic spontaneous tumors of the perineal skin and subcutis in female baboons have been reported and include cases of squamous cell carcinoma, lipoma, spindle cell sarcoma, myxoma, myxofibroma, myxosarcoma, and benign angiomyxoid proliferation.^7,8

Here we describe 2 cases of spontaneous malignant neoplasia within the sex skin of aged female chimpanzees. To our knowledge, this report represents the first documentation of sex skin neoplasia in chimpanzees.

Case Report

Animals.

All chimpanzees at the Michale E Keeling Center for Comparative Medicine and Research are maintained in group housing in accordance with the Animal Welfare Act¹ and the Guide for the Care and Use of Laboratory Animals¹⁵ and in a facility that is fully AAALAC-accredited. Chimpanzees have indoor–outdoor access to large outdoor habitat areas and indoor sheltered dens. The animals are fed a commercial diet (Chimpanzee Diet 7775, Harlan Teklad, Indianapolis, IN) twice daily; this high-fiber diet contains 20% protein, and the chimpanzees also receive 4 fresh-produce meals daily. Daily environmental enrichment is provided by using various food-puzzle devices, forage such as popcorn and sunflower seeds scattered in the grass of outdoor habitats, and manipulable items such as cardboard boxes and through rewards given during positive reinforcement training.

Case 1.

A 50-y-old female chimpanzee presented in November 2014 for a nonhealing wound to her sex skin (Figure 1 A). The wound had recently been traumatized, and a loose tissue tag was present at the wound site. Two biopsies were collected from the area; the wound was debrided, cleaned with dilute chlorhexidine (0.05%), and treated topically with a hydrocolloidal dressing (Derma-Gel, Veterinus, Ocala, FL) and bupivacaine HCl; and the animal was placed on enrofloxacin (5 mg/kg PO daily for 17 d). The primary differential diagnosis at the time of biopsy was a traumatic wound with secondary bacterial or fungal infection. This wound was first noted in January 2014, and at that time was treated with a dilute chlorhexidine rinse (0.05%) and sugar paste. The wound persisted until the next physical exam in June 2014 and was again treated with topical chlorhexidine rinse (0.05%) and sugar paste, and the animal was started on doxycycline (100 mg PO BID for 10 d). Although this wound was particularly persistent, this animal had a long history of trauma to her sex skin due to its unique pendulous anatomic structure.

Figure 1.

Case 1. (A) Ulcerated surface of a nonhealing sex skin wound that was later diagnosed as squamous cell carcinoma (November 2014). (B) An island of neoplastic cells exhibits intracellular bridging, marked anisocytosis and anisokaryosis, and frequent mitoses (arrow). Hematoxylin and eosin stain; bar, 50 μm. (C) Multifocally, neoplastic cells surround central accumulations of keratin (arrows). Hematoxylin and eosin stain; bar, 50 μm. (D) Progression of sex skin tumor growth (May 2015), with minimal tissue necrosis.

Biopsy sections were fixed in 10% buffered formalin, processed routinely for sectioning, and stained with hematoxylin and eosin. Both sections exhibited similar histologic characteristics. Expanding the dermis was an unencapsulated, poorly demarcated neoplasm composed of islands, nests, and trabeculae of neoplastic cells separated by variably dense fibrovascular stroma. Neoplastic cells were polygonal, with generally distinct cell borders, a small to moderate amount of vacuolated eosinophilic cytoplasm, and frequent prominent intercellular bridges. Nuclei were round to oval, with vesicular chromatin and 1 to 2 prominent nucleoli. Anisocytosis and anisokaryosis were marked, with occasional multinucleated cells (as many as 6 or 7 nuclei), and mitoses were 4 to 8 per high-power field, with frequent bizarre mitotic figures (Figure 1 B). Multifocally, individual neoplastic cells underwent keratinization and occasionally, islands and nests of neoplastic cells surrounded central accumulations of eosinophilic lamellar material (keratin) admixed with neutrophils, necrotic cell debris, and mixed bacterial colonies (Figure 1 C). Multifocally, the overlying epithelium was ulcerated, and the dermis adjacent to the neoplasm contained abundant mixed inflammatory cells (primarily lymphocytes with fewer histiocytes, eosinophils, and neutrophils). Neoplastic cells extended to the deep margin of resection. The histology of the mass was consistent with squamous cell carcinoma.

During an exam in January 2015, the animal's inguinal lymph nodes were mildly enlarged on palpation; bilateral lymph node biopsies were obtained to determine whether the tumor had metastasized. Histologic sections from both the right and left inguinal lymph nodes revealed no evidence of neoplastic cells. In addition, immunohistochemistry for cytokeratin (Clone AE1/AE3 Monoclonal Antibody, Ready to Use, Dako, Carpinteria, CA) was performed on the lymph node sections to confirm the absence of metastasis. At this time, the sex skin ulceration and tumor measured 6.0 cm × 5.5 cm and extended approximately 4 cm into the vaginal canal. Denser tumor tissue could be palpated 2 cm past the visual border of the superficial ulceration and tumor, and the mass appeared very vascular. Given the large size of the tumor, the sensitive nature of the region (vaginal and clitoral tissue), the high risk of postoperative infection from fecal contamination, and the animal's advanced age, the clinical veterinarians determined that she would not be a good candidate for tumor resection.

As of May 2015, the chimpanzee continues to be in good condition. The tumor has expanded deeper into the sex skin tissue but has shown minimal growth in diameter (Figure 1 D). The animal appears to be comfortable, and the tumor area is staying clean and has not been affected thus far with a secondary bacterial infection. The clinical veterinarians continue to monitor this animal very closely and have placed her on a quality-of-life watch, the details of which are described elsewhere.²⁰

Case 2.

A 48-y-old female chimpanzee presented in October 2014 for a nonhealing wound to her sex skin. The wound measured 4.9 cm × 4.5 cm, with a depth of approximately 2 cm. The wound was malodorous, although no discharge was discerned (Figure 2 A). Other relevant clinical findings included marked enlargement of the inguinal lymph nodes (Figure 2 B). The wound was cultured for both bacteria and fungi, 2 punch biopsies (diameter, 5 mm) of the lesion were obtained, and wedge biopsies were taken of the inguinal lymph nodes (one biopsy on each side). Clinically, the primary differential diagnosis was an infected wound, and the animal was started on cephalexin (500 mg PO TID).

Figure 2.

Case 2. (A) Ulcerated surface of a nonhealing sex skin wound that was later diagnosed as an adenocarcinoma. (B) Bilaterally, the inguinal lymph nodes are enlarged (arrows).

Multiple sections from the mass were processed routinely and examined histologically. The dermis of the nonhaired skin contained an unencapsulated, expansile, and invasive neoplasm composed of sheets, nests, and cords of neoplastic cells on a moderate amount of fibrovascular stroma (Figure 3 A). Neoplastic cells were polygonal, with generally distinct cell borders and a small to moderate amount of eosinophilic cytoplasm (Figure 3 B). Nuclei were round and vesicular, with 1 to 3 prominent nucleoli, and there were occasional multinucleated neoplastic cells. Anisocytosis and anisokaryosis were moderate to marked, and mitoses were 3 to 7 per 400× field and occasionally bizarre. There were small to moderate numbers of lymphocytes and plasma cells within the adjacent dermis and subcutis. Neoplastic cells extended to the deep margin and multifocally to the lateral margins of resection. In addition, the normal architecture of both the right and left inguinal lymph nodes was effaced by the neoplastic population described above, admixed with abundant collagenous tissue (scirrhous response).

Figure 3.

Case 2. (A) The dermis and subcutis of the sex skin contains an unencapsulated, expansile and invasive neoplasm. Hematoxylin and eosin stain; bar, 250 μm. (B) The neoplasm is arranged in sheets, nests, and cords of polygonal neoplastic cells. Hematoxylin and eosin stain; bar, 50 μum. (C and D) Neoplastic cells exhibit diffuse strong positive cytoplasmic staining for (C) cytokeratin and (D) vimentin. Bar, 250 μm.

At this time, the mass was diagnosed as a metastatic malignant neoplasm, given that the histologic features were not indicative of a specific tumor type. To further characterize the neoplasm, immunohistochemistry was performed on sections from both the primary tumor and the left inguinal lymph node. Diffusely, all neoplastic cells exhibited strong positive cytoplasmic staining for both cytokeratin (Clone AE1/AE3 Monoclonal Antibody, Ready to Use, Dako; Figure 3 C) and vimentin (Clone V9 Monoclonal Antibody, Ready to Use, Dako; Figure 3 D). Neoplastic cells were diffusely negative for S100 (Polyclonal Antibody, Ready to Use, Leica Biosystems, Buffalo Grove, IL), CD31(Clone 1A10 Monoclonal Antibody, Ready to Use, Leica Biosystems), and CD45 (Clone 2B11+PD7/26 Monoclonal Antibody, Ready to Use, Dako).

In light of the histologic and immunohistochemical features, the preferred differential diagnoses at this time included anaplastic carcinoma and amelanotic melanoma, as well as consideration that the sex skin mass was a metastatic focus from an unidentified primary location elsewhere in the body. The case was then referred to the Joint Pathology Center for consultation. The diagnosis favored by the Joint Pathology Center was metastatic adenocarcinoma, likely derived from apocrine glands. Amelanotic melanoma was ruled out by negative immunohistochemistry results for Melan A and HMS45 staining performed at the Joint Pathology Center. In addition, the consultant noted that in view of the presence of metastasis and the aggressive cellular features of the neoplasm, the prognosis for the animal was extremely guarded.

Treatment with a dilute chlorhexidine spray (0.05%) was initiated to help keep the tumor surface clean from debris and prevent secondary infection. In December 2014, it appeared that the tumor had become painful, and the animal no longer tolerated its cleaning. A recheck examination showed the tumor had increased in size to 6.2 cm × 7.0 cm and extended approximately 7 cm into the vaginal orifice. In addition, the inguinal lymph nodes had become severely enlarged (left, 6.3 cm × 6.6 cm; right, 8.0 cm × 8.4 cm). The sex skin wound associated with the tumor exhibited necrosis with associated purulent discharge. Because of quality-of-life concerns, euthanasia was elected on 19 December 2014.

Necropsy confirmed the presence of a malignant neoplasm in the left portion of the sex skin, both inguinal lymph nodes, and the right caudal abdomen. The sex skin tumor was ulcerated and reddened with brown scabs. Both inguinal areas had large subcutaneous masses (4 cm × 2 cm × 2 cm) that were firm, pale tan, and multilobulated; no normal lymph node tissue was identifiable. The mass in the abdomen was firm, pale tan, multilobulated (7 cm × 5 cm × 4 cm; Figure 4 A) and adhered to the caudal abdominal wall adjacent to the right inguinal mass. Microscopic examination of the sex skin mass, inguinal lymph nodes and adjacent tissue, and mass in the right caudal abdomen revealed neoplasms with histologic features consistent with the earlier biopsy (Figure 4 B).

Figure 4.

Case 2. (A) The abdominal mass is firm, multilobulated, and adherent to the right caudal abdominal body wall. (B) Cellular features of the abdominal mass are consistent with those of the neoplastic populations within the sex skin and inguinal lymph nodes. Hematoxylin and eosin stain; bar, 50 μm.

Discussion

This report describes 2 cases of malignant sex skin neoplasia in aged female chimpanzees. Although the development of neoplasia in chimpanzees previously was considered to be an uncommon occurrence, populations of aged captive chimpanzees are living beyond the previously hypothesized lifespan for the species.²⁵ With this longevity comes the increased incidence of age-related chronic diseases and neoplasia.²⁵

The mass in case 1 was diagnosed as a squamous cell carcinoma (SCC), a malignant epithelial tumor originating in the epidermis or squamous mucosal epithelium. SCC was commonly reported in a survey of 11 zoos and NHP facilities, with cases in the skin, oral cavity, stomach, esophagus, lung, cervix, vagina, and penis of macaques, as well as within the skin, oral cavity, and perineum or sex skin of baboons.^13,24,29 In both humans and NHP, the occurrence of SCC has been associated with chronic irritation, carcinogens, prolonged sunlight exposure and papillomavirus infection.^13,24,29 In baboons, in addition to chronic trauma (nonhealing fight wounds) and sun exposure, chemical disinfectants and perineal infection with Herpesvirus papio 2 have been proposed to be involved in the development of SCC.^13,24,29 Because cycles of anogenital swelling may continue until death in captive chimpanzees, there are repeated opportunities for trauma and wounding to the delicate sex skin, particularly during times of maximal swelling.^2,19,26,35 The 50-y-old female chimpanzee presented as case 1 had a long history of repetitive trauma to her sex skin. This trauma was due in part to the unique pendulous anatomy of her sex skin, which served as a target for injury during aggressive social interactions, and likely predisposed her to the neoplasia observed later in her life. Given her advanced age (50 y) and continuous access to outdoor habitat areas with long durations of time spent outdoors, prolonged sunlight exposure likely also contributed to the development of neoplasia.

In the perineal region of NHP, SCC often presents as a chronic, ulcerated, nonhealing wound and is frequently initially misdiagnosed as trauma, a common occurrence on the sex skin.^13,29 Indeed, the animal in case 1 originally presented for an open wound on the sex swelling that was not healing well. With the progression of disease, the neoplasm can invade locally into adjacent soft tissues and bone or metastasize, particularly to regional lymph nodes.^13,29 In a report of 13 cases of SCC in baboons, 4 cases involved the sex skin, and 3 of the cases initially presented as skin laceration or sloughing.¹³ Because of disease progression (local invasion in all cases, and bone and lymph node involvement in one animal each), all of the baboons were euthanized within 10 mo of presentation.¹³ In the chimpanzee in case 1, both regional (inguinal) lymph nodes were examined histologically and immunohistochemically for metastasis but were not affected. However, because the neoplastic population did extend to the deep margin of excision, she is frequently and closely monitored for local and distant metastatic foci.

Treatment options for sex skin neoplasia can vary based on the tumor type. In considering the SCC diagnosed in case 1, a common veterinary therapeutic option is the use of topical agents, such as 5-fluorouracil, a topical chemotherapeutic with direct cytotoxicity. Unfortunately, the burning sensation associated with this medication likely would deter the animal from voluntarily cooperating with the application process. In addition, chimpanzees would likely wipe away the medication from the target tissue and ingest it. Ingestion could damage normal mucosal cells, such as those found in the mouth which are particularly susceptible to the toxic effects of 5-flurouracil. Given these risks and natural chimpanzee behavior, the clinical veterinarians determined that topical chemotherapeutics would not be a safe treatment option for case 1. Surgical resection was considered but was rejected in view of the large area that needed to be excised, the sensitive nature of the vaginal and clitoral tissue to be resected, the risk of fecal infection of the postoperative surgical site, and the advanced age of the animal. In this particular case, the potential harm that might result from surgical resection was deemed to outweigh the potential benefits of the surgery. Ultimately the clinical team elected to continually monitor this animal for quality of life and to conservatively treat her with topical cleansing of the tumor area and pain-alleviating medications as required.

Case 2 presented a significant diagnostic challenge. The chimpanzee originally presented for a wound on the sex skin that was not healing despite antibiotics and topical treatments. However, histologic examination of biopsies of the mass and draining lymph nodes led to the diagnosis of a malignant neoplasm that had metastasized to both inguinal lymph nodes. At this time, it also was considered possible that the mass on the sex skin was not a primary tumor and that this animal might harbor additional neoplastic foci (including a primary focus). Immunohistochemical staining was then performed to further characterize the tumor. Negative results for multiple immunohistochemical stains (S100, CD31, CD45, Melan A, and HMS45) ruled out tumors of neuroendocrine and round-cell origin, hemangiosarcoma, and melanoma. Interestingly, however, neoplastic cells exhibited positive staining for both cytokeratin (an epithelial marker) and vimentin (a mesenchymal marker). Historically, cytokeratin and vimentin were used to distinguish epithelial from mesenchymal origin, with few tissues known to normally express both intermediate filaments.³ However, more recent studies have indicated that there is an expanding list of neoplasms that may exhibit positive staining for both cytokeratin and vimentin. In animals, this unique pattern of staining has been associated with anaplastic carcinomas, mesotheliomas, amelanotic melanomas, renal carcinomas, canine prostatic carcinoma and hepatocellular carcinoma, and feline bronchogenic adenocarcinomas.^4,22

In addition, poorly differentiated neoplastic polygonal cells can retain vimentin immunoreactivity during epithelial-to-mesenchymal transition, as may have occurred in case 2. Epithelial-to-mesenchymal transition converts epithelial cells into mesenchymal cells, a cellular program first identified in the 1980s.^17,32,36,37 This process is considered normal during implantation, embryogenesis, and wound repair but is often implicated in epithelial cancer progression and metastasis.^17,32,36,37 Epithelial-to-mesenchymal transition requires complex changes in cell architecture and behavior and accordingly, many molecular processes are involved in the transition.^17,32,37 Cells that have undergone epithelial-to-mesenchymal transition lose characteristics typical of epithelial cells (cell shape, apical basal polarity, and expression of the cell adhesion molecule and epithelial marker E-cadherin) and can then become spindle-shaped and express proteins characteristic of mesenchymal cells, including vimentin, smooth muscle actin, desmin, fibroblast-specific protein 1, fibronectin, and stromelysin 3.^{17,32,33,36,37}

The final diagnosis for case 2 was an anaplastic carcinoma with resemblance to apocrine adenocarcinoma. The primary site of the mass is unclear currently. Although chimpanzees do not have anal sacs, they do have anal glands, so perhaps this lesion represents localized spread from a neoplasm of the anal glands. Cases of apocrine carcinoma in NHP have been reported only rarely and include a papillary apocrine carcinoma in the right pectoral region of a 19-y-old male capuchin monkey and a low-grade apocrine adenocarcinoma in the thoracic region near the right mammary gland of an 8-y-old male marmoset.^5,18 Although adenocarcinomas derived from the apocrine glands of the anal sac have been associated with a paraneoplastic syndrome of hypercalcemia in dogs, at the time of euthanasia, the chimpanzee's calcium level was within normal limits (9.1 mg/dL; reference range, 8.1 to 10.2 mg/dL).²³ In humans, apocrine carcinomas are rare neoplasms that usually arise from axillary glands, although other sites, including the eyelid, ear, scalp, anogenital region, and breast are also possible.¹² These neoplasms can exhibit both local (regional lymph nodes) and distant (lung, bone) metastasis.¹² In addition, apocrine carcinomas have developed from the apocrine sweat glands of the vulva in humans, although no similar cases have been reported for NHP.¹⁶ In humans, these neoplasms also have potential for regional and distant metastasis.¹⁶

Because the sex skin is a target organ for ovarian steroids, it contains receptors for both estrogen and progesterone.^11,27 Although swelling has been correlated with fluctuations in ovarian steroid hormones (estrogen and progesterone), the mechanism of hormonal regulation has not been completely determined.^{10,14,27,30,35} Whereas early research suggested that sex swelling was controlled by estrogen alone, recent reports imply control either by the ratio of progesterone to estrogen or by progesterone alone.³⁵ Estrogen is known to dominate the follicular phase (maximal tumescence), whereas during the luteal phase, progesterone has been implicated in detumescence, through the induction of localized estrogen withdrawal and downregulation of estrogen receptors.^{11,19,27,34,35} Interestingly, neoplastic cells from the sex skin mass in case 2 were diffusely negative for both estrogen (Clone SP1 Monoclonal Antibody, Ready to Use, Ventana Medical Systems, Tucson, AZ) and progesterone receptor (Clone 1E2 Monoclonal Antibody, Ready to Use, Ventana Medical Systems) expression.

As with case 1, a potential treatment option for the sex skin adenocarcinoma of case 2 was complete surgical excision of the tumor with wide margins. However, consideration was given to the advanced tumor stage and the presence of metastasis. Additional concerns regarding this animal included her advanced age and the risk of potential postoperative surgical complications. Chimpanzees, in particular, present additional surgical challenges, in that they often attempt to remove sutures postoperatively and may contaminate postoperative areas with fecal matter. In addition, social conspecifics may be interested in grooming (and possibly traumatizing) the postoperative area, thereby necessitating individual housing of the affected animal—an undesirable situation for social chimpanzees. Given the presence of local metastases in case 2, surgical resection was not considered to be a viable treatment.

Both animals in this report were advanced in age (50 and 48 y) and had lived in habitats with both indoor and outdoor access. The hypopigmented sex skin had been exposed to prolonged sunlight throughout the lifespans of these animals, likely predisposing case 1 to an increased risk of SCC. In addition, the sex skin of chimpanzees is often targeted during aggressive social interactions, and repetitive tissue trauma has been shown to play a role in the development of neoplasia.^13,29 As the captive chimpanzee population continues to age, the reported incidence of not only sex skin neoplasms, but also other skin neoplasms, is likely to increase, especially in populations that are housed in a natural outdoor habitat.