Polyostotic Fibrous Dysplasia in a Cynomolgus Macaque (Macaca fascicularis)

Abstract

A 2.3-y-old female cynomolgus macaque (Macaca fascicularis) presented with a broken right tibia and fibula. Radiographs showed multiple cyst-like defects in all long bones. We suspected that both fractures were pathologic because they occurred through these defects. Ultrasonography, MRI, and dual X-ray absorptiometry revealed that the defects were filled with soft tissue. Grossly, the bones were abnormal in shape, and a gelatinous material filled the defects and the surrounding marrow cavity. Histologically, the gelatinous material was composed of fibrin and cartilage; few normal bone cells were seen. Genetic testing revealed extra material on the short arm of chromosome 8 in all tissues examined, but no copy number alterations of likely clinical significance were observed, and no abnormalities were found that were unique to the lesions. In light of the clinical signs and radiographic and pathologic findings, polyostotic fibrous dysplasia was diagnosed. This report represents the first documented case of fibrous dysplasia in a cynomolgus macaque.

Cynomolgus macaques (Macaca fascicularis) are small nonhuman primates often used in research. Female cynomolgus macaques have an average weight of 2.5 to 5.7 kg, live for approximately 37 y in the wild, and typically reach sexual maturity at approximately 52 mo (4.3 y) of age.²⁵ They are highly adaptable social animals that form large groups in which juvenile and young macaques play frequently with conspecifics of similar age and sex.

Case Report

A 3.0-kg, 2.3-y-old female cynomolgus macaque presented to the clinic for nonweightbearing in her right leg. She was housed in a large indoor–outdoor breeding colony that is in compliance with federal regulations governing the use of animals in research and with AAALAC guidelines. The macaque was housed and all procedures were performed under an IACUC-approved protocol. All examinations of the animal were conducted under sedation with ketamine (10 mg/kg IM; Ketamine Hydrochloride Injection, Bioniche Teoranta, Lake Forest, IL). Routine radiographs taken after physical examination revealed a transverse fracture through both the tibia and fibula. Multiple regions of irregular radiographic bone density with cystic and sclerotic areas were noted in the long bones included in the radiograph (tibia, fibula, and femur). The fractures occurred at the sites of some of these irregularities. The macaque was placed in a cast and put on cage rest. Differential diagnoses at that time included fibrous dysplasia, osteogenesis imperfecta, bone cysts, osteomyelitis, and neoplasia.

Blood work (CBC, complete chemistry) was unremarkable. ALP levels were within normal limits, as was the calcium:phosphorus ratio. The macaque tested seronegative for B virus (Cercopithecine herpesvirus 1), SIV, and simian T-cell leukemia virus and seropositive for simian retrovirus.

Thirteen days after initial presentation, additional radiographs were taken of all the long bones (Figure 1), as well as the chest, abdomen, and skull. Multifocal regions of fairly well-defined, osteolytic, occasionally expansile lesions with adjacent sclerosis were present in all long bones: the distal third of the right radius and ulna; middle third of the left radius and ulna; both distal humeri; distal half of the left tibia and fibula; proximal and distal third of the right tibia and fibula; distal half of both femurs; and both femoral necks, with the right being more severely affected. No lesions were noted in the skull, ribs, spine, or phalanges. The fracture sites were healing at a normal pace. The cast was replaced and the macaque allowed further cage rest.

Figure 1.

Radiographs of affected Macaca fascicularis at initial presentation. (A) Left arm. (B) Right arm. (C) Left leg. (D) Right leg. (E, F) Pelvis and legs. Note abnormal lucencies (arrows) in all long bones. A fracture is evident in panels D and F (under the R marker).

The radiographs were reviewed by a veterinary radiologist (AV). There were oval to rounded, cystic, radiolucent regions of lysis involving the diaphyses and metaphyses of the long bones, with faint trabecular markings within these lytic zones. These lytic regions were well-demarcated, had a multilobulated appearance, and were slightly expansile, as evidenced by variable thinning of the cortices and slight widening of the diaphyses at these sites. Medullary sclerosis of the diaphyses, appearing as multifocal sclerotic and lucent regions, was present. The lesions involved the long bones, with the vertebrae and physes appearing to be spared. The findings were most consistent with multicentric bone cysts. Other differential diagnoses included metabolic-type disease and osteogenesis imperfecta, both of which were considered unlikely but could not be excluded.

The macaque was checked for healing routinely, with cast changes approximately every 2 wk. When the fracture was manipulated, the animal received pain medication (30 mg PO twice daily; Children's Motrin Berry Flavor, Johnson and Johnson, New Brunswick, NJ) for 3 to 8 d as needed based on presentation of signs associated with pain. When the fracture had healed completely, the macaque was returned to group housing for observation. She was placed in a calm, small group of 6 macaques that were similar to her in age. Bone biopsies were considered but not completed, because of the fragile nature of her bones and potential for another fracture.

In group housing, the macaque did well; she was able to locomote and interact normally with her cagemates. Approximately 3 mo later, the macaque again presented because of nonweightbearing in her right leg. Physical examination was performed, and radiographs were taken (Figure 2). A fracture was noted at the right femoral neck, through the site of a previously noted bony resorptive lesion. An Ehmer sling was placed, and advanced imaging and euthanasia were scheduled. The macaque did well in the Ehmer sling and began to heal; at no time did she lose weight or become inappetant. There were no behavioral or physical indicators of pain or distress while the macaque was in the Ehmer sling. After the second application of the Ehmer sling, the macaque removed the bandage. No sign of pain was present, and because she was not using her leg, the bandage was not replaced.

Figure 2.

Radiograph taken at second presentation. Note fracture in right femoral neck.

On the day of euthanasia, physical examination revealed decreased range of motion in the right (fracture-side) hip and knee, with muscle contracture. All other parameters were within normal limits. The affected macaque was transported for MRI using several types of weighted scans (T1, T2, fat-specific). Multiple lesions were noted in the long bones. The lesions were not fluid- or fat-filled but had the same density as soft tissue. A full necropsy was performed. All diagnostic tests and necropsy were completed within approximately 3 to 4 h of euthanasia.

At necropsy, each long bone contained both normal and abnormal areas (Figure 3). The bones were distorted at sites concurrent with the radiographic lesions, usually evidenced by thickening of the diaphysis and mild bowing. The marrow cavity was filled with a gelatinous substance, and the cortices were thinner than normal. The only other abnormal finding was that the right ovary was enlarged to approximately 5 times normal size and cystic. Tissue samples were taken for histologic evaluation, fixed in 10% neutral buffered formalin, decalcified, processed conventionally, embedded in paraffin, cut at 5 µm, stained with hematoxylin and eosin, and evaluated by light microscopy by a board-certified veterinary pathologist (EJD). There were similar changes throughout all the long bones examined. The cortex was generally thin and blended into a zone of bony trabeculae lined by few osteoblasts and separated by abundant vascularized fibrous connective tissue composed of densely packed fibroblasts in a collagen matrix. This fibroblastic proliferation extended into and filled the marrow cavity. Some sections also contained irregular islands of disorganized hyaline cartilage either within the areas of fibrosis or uniformly replacing all the subcortical structures and marrow cavity (Figure 4). The differential diagnoses included polyostotic fibrous dysplasia and fibrous osteodystrophy (nutritional hyperparathyroidism), which was considered unlikely because there was absence of parathyroid gland hyperplasia. The ovary had scattered mature follicles and organizing corpora luteum. The final diagnosis was determined to be polyostotic fibrous dysplasia, in light of the clinical history and radiographic and pathologic findings.

Figure 3.

Long bones from (A through C) the affected monkey and (D through F) an age-matched control. (A) Right femur of affected macaque. Note the swelling and loss of normal cortical contour (femoral head proximal to fracture site not included). (B) Right tibia and fibula of affected macaque. Note the swelling and bowing at the initial fracture site. (C) Cross-sections through the right femur of the affected macaque. Note the swollen area with abnormal (gelatinous) marrow cavity and other relatively normal bone. (D) Right femur of control animal. (E) Right tibia and fibula of control animal. (F) Cross-sections through right femur of control macaque.

Figure 4.

Histologic appearance of long bones from affected monkey. (A) Right femur. Note the thin cortex, decreased trabeculae, and medullary replacement by fibrous and cartilaginous tissue. (B) Right fibula. Note the complete replacement of the medullary cavity by cartilage.

Clonal chromosome aberrations have been reported in human cases of fibrous dysplasia.⁵ The immediate lineage of this macaque was known, with the sire, dam, and one male half-sibling from the same dam being the only directly related individuals. The dam and half-sibling had no history of abnormalities and were normal on physical examination. The half-sibling had radiographically normal bones. The sire was unavailable for physical examination but had no history of abnormalities.

Cytogenetic studies were performed on the affected macaque, dam, and half-sibling. Two aspirates from affected bone (left radius and left ulna) and a skin sample from the back of the affected animal were obtained and cultured. Heparinized peripheral blood samples were obtained from the affected macaque, dam, and half-sibling. The blood samples were cultured in RPMI-1640 supplemented with 15% FBS, 1% L-glutamine, 1% antibiotic–antimycotic solution (Sigma-Aldrich, Atlanta, GA), 1.5% concanavalin A, and 1.5% pokeweed mitogen and harvested in 3 d. The skin and bone cyst aspirates were treated briefly with antimicrobial and antifungal agents and then placed in culture by using standard explant and collagenase treatments. The tissues were cultured in Minimal Essential Medium (Invitrogen, Carlsbad, CA) supplemented with L-glutamine, FBS, antibiotics, and antimycotics and incubated at 37 °C in a 5% CO₂ environment. Cells were harvested and G-banded by using a trypsin–Giemsa staining technique. Twenty well-spread, high-resolution metaphases from each sample were analyzed. Karyotypes were arranged according to size, following the nomenclature of Cambefort and colleagues.³ All the cells in each tissue from the affected animal had 42 chromosomes, the normal number for macaques. Each cell also had a small amount of extra chromatin on the tip of the short arm of one chromosome 8 (which is homologous to human chromosome 8) that was not seen in the dam or half-sibling's karyotypes.

DNA samples from the affected animal and a sex-mismatched normal cynomolgus control were prepared for array comparative genomic hybridization (Microarray Laboratory, University of Utah) using the Human Genome CGH Microarray 44K platform (amadid 14950; Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocols. The data were analyzed by using CGH Analytics 3.5 software (Agilent Technologies) using the ADM-2 algorithm. This analysis showed a clear duplication for the entire X chromosome, which was expected in light of the sex-mismatched control. The derivative log ratio was 0.56, which was higher than that routinely seen in clinical studies of human DNA (typically less than 0.2), but this value was acceptable for this interspecies comparison, and the data were generally without background noise. For the autosomes, one small duplication (approximately 50 kb) was detected on human chromosome 20, which contains the zinc finger protein 337 gene (ZNF337). No other abnormal copy number alterations were observed.

Discussion

Fibrous dysplasia is an uncommon skeletal condition in which normal bone is replaced with fibro-osseous tissue and has a widely varied presentation. The term was initially coined by Louis Lichtenstein in 1938.¹⁶ It is a disorder of development, with no known heritable link⁴ or sex predisposition.^10,28 Lesions can be found in a single bone (monostotic) or, less often, several bones (polyostotic), as in the current case. McCune–Albright syndrome is the most severe form and is characterized by polyostotic fibrous dysplasia plus an endocrinopathy (often precocious puberty) or cutaneous hyperpigmentation (café au lait spots) or both.^{1,4,7,10,14,15,17,23} It is difficult to determine whether the cynomolgus macaque presented here was hyperpigmented, given that their skin is more pigmented naturally than that of humans, making the differentiation of café au lait spots difficult. Despite the macaque's young age, she did have an active ovary with evidence of ovulation, but whether she had begun functional estrus cycles is unknown, making it unclear whether she exhibited precocious puberty.

Fibrous dysplasia can present with varied severity and often goes undiagnosed unless a complication, such as a fracture, arises. Pathologic fractures of weight-bearing limb bones are the primary cause of morbidity, with the 2 sites most commonly affected being the femur and the skull base.^1,15 Fibrous dysplasia is most commonly diagnosed in young patients, with the first symptom appearing before the age of 15 y in 80% of known human cases.⁴ Most lesions are detected by the age of 30.^7,10,28 Some literature reports that the peak fracture rate is within the first decade of life.^7,12,14,15 As patients complete puberty, lesions usually stop progressing and remain stable.^9,21

Radiographically, fibrous dysplasia can present with a number of different appearances. The most common radiographic finding is a ‘ground-glass’ appearance.^1,7,9,15,28 In the polyostotic form, the long weight-bearing bones often exhibit bowing due to weakness and microstress fractures over time.²¹ Lesions in long bones often present with a well-defined sclerotic border (‘ring sign’).¹⁰ The lesions usually are contained within the medullary canal; however, they can arise in both cancellous and cortical bone, leading to deformation of the bone.⁷ An important differential to consider is ossifying fibroma, which can present clinically similarly, but is more aggressive in nature. Ossifying fibroma lesions almost always begin in the cortex,¹⁸ unlike fibrous dysplasia. The age at radiographic diagnosis is a predictor of the appearance of lesions and whether secondary changes are present, such as aneurismal cysts. MRI can be a useful tool to diagnose aneurismal bone cysts.¹⁵ The lesions in this macaque had a varied presentation, with some cyst-like lesions, thinning of the cortices, and a ground-glass appearance. The lesions seemed to be confined to the medullary canal, with some distortion of the cortical bone.

Grossly, bones with fibrous dysplasia are described as having a yellowish discoloration with a gritty feel.⁷ This manifestation is due to the disorganized trabeculae within the lesions. On cut surface, the gross lesions in this macaque were pale tan in color, with a gelatinous feel. Individual bones contained both grossly normal and abnormal sections.

Histologically fibrous dysplasia presents with a ‘Chinese character’ appearance of abnormal disorganized trabecular bone.¹⁵ The woven bone is immature, devoid of rimming osteoblasts and osteoclasts, with spindle cells in a whorled arrangement.¹⁰ The trabeculae show a pattern of disorganized deposits, despite thickness and maturity under polarized light.^7,10,23 Normal maturation of bone follows the pattern of woven bone deposition followed by rimming of spicules with rows of osteoblasts which then deposit bone matrix layers (osteiod). Over time, the woven bone is replaced by lamellar bone.²³ Fibrous dysplasia lesions exhibit no rimming of trabeculae by osteoblasts and no evidence of lamellar replacement of woven bone. Ossifying fibroma differs in that the trabeculae are surrounded by active osteoblasts.^13,18,27 In fibrous dysplasia, often the cortical bone is preserved but is thin,^23,28 as was seen in the current case. Marrow spaces in fibrous dysplasia lesions are filled with cellular tissues arranged in a random fashion.²³ Many human cases have active fibroblasts.²¹ Histologic changes due to treatment may complicate obtaining an accurate diagnosis.

Malignancy in fibrous dysplasia is rare, with osteosarcoma being the most common neoplasm.^1,7,10,15,28 Polyostotic forms of the disease are more likely to develop malignancy than are monostotic forms.^10,28 Other secondary changes include aneurismal bone cysts at the sites of intralesional hemorrhage.¹⁰ Aneurismal bone cysts were a consideration for this macaque and may have been present as secondary changes due to fibrous dysplasia.

Diagnosis of fibrous dysplasia primarily is based on clinical findings, radiographic evidence, and histologic findings.^4,15 Diagnostic laboratory results may also play a role, with elevation of markers of bone turnover (ALP, calcium:phosphorus ratio, and so forth) and the presence of other derangements from normal (hypophospatemia, phosphaturia, endocrine abnormalities). Serum biochemistry results for this macaque were unremarkable, and a urinalysis was not obtained.

The only treatment available for fibrous dysplasia is to manage pain and repair fractures. In human medicine, the fractures often are pinned. Compared with the monostotic form, polyostotic fibrous dysplasia has a higher risk of deformity and complications, due the presence of multiple lesions. In addition, bisphosphonates to inhibit osteoclasts are used in human medicine, with some reported success.^4,7,15 Because our macaque was bred as part of a research colony and would be unsuitable for study, she was euthanized. While diagnostics were being performed, she was treated with pain medication, and the fractures were treated accordingly.

Fibrous dysplasia is a rare disorder in humans and appears to be equally rare in animals. There are only 2 reports of fibrous dysplasia in nonhuman primates, both occurring in the maxilla; one case involved a spider monkey¹¹ and the other a cebus monkey.²⁹ Another reported case of multiple cystic lesions in the bones of a spider monkey was determined to be Gorham–Stout syndrome.³⁰ Fibrous dysplasia has also been noted to occur in a canine⁸ and greater kudu.²⁹

New advances in knowledge about fibrous dysplasia have pointed to skeletal stem cell mutations in the imprinted GNAS gene on chromosome 20q13 that mediates the pathologic cystic changes in bone.^4,7,15,17,24 This mutation leads to abnormal proliferation and differentiation of bone marrow stromal cells. The stage of development at which the mutation occurs seemingly dictates the severity of the signs seen.^4,15 The GNAS mutation is likely to be present in significant numbers only in the affected tissues.¹⁵

Comparative studies on baboon and macaque chromosomes have shown they have virtually identical karyotypes and a close relationship to the human karyotype,^2,20 such that human DNA probes can be used successfully to identify baboon and macaque chromosomal abnormalities.^19,26 The ZFP337 gene, which is located within the 50-kb duplication seen in the affected macaque, is a highly conserved gene that is expressed in multiple tissues across species. This gene does not appear to be associated with any abnormal phenotype and is included in a genomic region containing copy number variants (UCSC Genome Browser, http://genome.ucsc.edu), suggesting that the duplication found in the affected animal is a normal variant. In addition, a gain in this region has also been reported in an otherwise healthy person in the Database of Genomic Variants (http://projects.tcag.ca/variation/). These data, combined with the sex chromosome control data, indicate that there were no copy number alterations of likely clinical significance in the affected macaque.

The finding of clonal chromosome changes in several reports of fibrous dysplasia indicates that this condition may, at least in some cases, be neoplastic in nature.^5,6,22 Trisomy 2 and structural 12p13 aberrations have been reported as recurring abnormalities. Although the macaque we report may have had a constitutional chromosomal variant or hemicryptic translocation, none of the abnormalities were unique to the lesions.