www.elsevier.com/locate/humpath

Human Pathology (2014) xx, xxx–xxx

Original contribution

USP6 gene rearrangements occur preferentially in giant cellreparative granulomas of the hands and feet but not ingnathic location☆

Narasimhan P. Agaram MBBSa,⁎, Francois V. LeLoarer MDa, Lei Zhang a,Sinchun Hwang MDb, Edward A. Athanasian MDc, Meera Hameed MDa,Cristina R. Antonescu MDa,⁎

aDepartment of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065bDepartment of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065cDepartment of Orthopedic Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, 10065

Received 3 December 2013; revised 15 January 2014; accepted 24 January 2014

a

0h

Keywords:USP6;Giant cell reparativegranuloma;

Solid ABC

Summary Giant cell reparative granulomas (GCRGs) are lytic lesions that occur predominantly in thegnathic bones and occasionally in the small bones of the hands and feet. They are morphologicallyindistinguishable from, and are regarded as synonymouswith, solid variant of aneurysmal bone cysts (ABC)in extragnathic sites. Identification of USP6 gene rearrangements in primary ABC has made possibleinvestigating potential pathogenetic relationships with other morphologic mimics.USP6 gene alterations ingiant cell–rich lesions (GCRG/ABC) of small bones of the hands and feet have not been previously studied.We investigated USP6 gene alterations in a group of 9 giant cell–rich lesions of the hands and feet andcompared the findings with morphologically similar lesions including 8 gnathic GCRGs, 22 primary ABCs,8 giant cell tumors of bone, and 2 brown tumors of hyperparathyroidism. Overall, there were 49 samplesfrom 48 patients including 26 females and 22 males. Of the 9 lesions of the hands and feet, 8 (89%) showedUSP6 gene rearrangements, whereas no abnormalities were identified in the 8 gnathic GCRGs, 2 browntumors, or 8 giant cell tumors of bone. Of the 22 primary ABCs, 13 (59%) showed USP6 generearrangements. In conclusion, most GCRGs of the hands and feet represent true ABCs and should beclassified as such. The terminology of GCRG should be limited to lesions from gnathic location.Fluorescence in situ hybridization forUSP6 break-apart is a useful ancillary tool in the diagnosis of primaryABCs and distinguishing them from GCRGs and other morphologically similar lesions.© 2014 Published by Elsevier Inc.

1. Introduction

☆ Disclosure/Conflict of Interest: None.⁎ Corresponding authors.E-mail addresses: agaramn@mskcc.org (N. P. Agaram),

ntonesc@mskcc.org (C. R. Antonescu).

046-8177/$ – see front matter © 2014 Published by Elsevier Inc.ttp://dx.doi.org/10.1016/j.humpath.2014.01.020

Giant cell reparative granuloma (GCRG) was firstdescribed by Jaffe [1] in 1953 as a benign nonneoplasticprocess related to intraosseous hemorrhage and limited tothe gnathic sites, either mandible or maxilla. In 1962,Ackerman and Spjut [2] reported 2 lesions involving thephalanges, which they termed giant cell reaction, defined

2 N. P. Agaram et al.

as “a rare, benign, non-neoplastic lesion involving smallbones of the hands.” In 1980, Lorenzo and Dorfman [3]published the first large series of 8 cases of “giant cellreparative granulomas of the short bones of the hand andfeet” and reported their recurrence potential, with 4 of8 lesions recurring 1 or more times. They also noted themorphologic similarity to other giant cell–rich lesions, suchas aneurysmal bone cyst (ABC) and GCRG of the mandibleand maxilla, and hypothesized that these lesions are relatedresponses to intraosseous hemorrhage. Gnathic GCRGshave been also termed as central giant cell lesions or cen-tral giant cell granulomas.

The terminology of solid aneurysmal bone cyst wasintroduced by Sanerkin et al [4] in 1983, who reported 4cases of non-cystic lesions involving bone (3 in the spine and1 in the ethmoid), with morphologic features similar to thatseen in ABC. They also noted the histologic overlap withother giant cell–rich lesions, including GCRG. Ratner andDorfman [5], in 1990, reported 20 additional cases of GCRGof hands and feet and reemphasized the difficult distinctionof solid areas of ABC from GCRG. Subsequently, Bertoni etal [6], Oda et al [7], and Ilaslan et al [8] reported lesions withsimilar morphology in the long bones and designated them assolid ABC or extragnathic GCRG.

Cytogenetic abnormalities in ABC were first reported byPanoutsakopoulos et al [9] in 1999, with a recurrentchromosomal translocation t(16;17)(q22;p13) being identi-fied in 2 cases. Subsequently, Dal Cin et al [10] reported 2additional cases of solid and extraosseous ABC with thesame translocation. In 2004, Oliveira et al [11] identifiedthe fusion gene partners as CDH11 (osteoblast cadherin 11),on chromosome 16q22, and USP6 (ubiquitin protease 6,aka Tre2 oncogene), on chromosome 17p13. TheCDH11-USP6 fusion transcript was identified only inprimary ABC but not in secondary ABC [12]. Subse-quently, additional variant translocations were identified inABC, with USP6 being the common gene partner, whosetranscription is up-regulated by promoter swapping withother genes, including ZNF9, COL1A1, TRAP150, andOMD [13].

Because GCRG and solid ABC cannot be distinguishedmorphologically, we sought to investigate if USP6 geneticalterations, a hallmark of primary solid and classic ABC, arealso a feature of GCRG occurring either in the gnathic orsmall bones of the hands and feet.

Table 1 BAC Clones for USP6 gene

Clones Cytoband Gene GP-S GP-E

RP11-111I16 17p13.2 T-USP6 4824242 4968438RP11-81A22 17p13.2 T-USP6 4639600 4838930RP11-457I18 17p13.2 C-USP6 5152461 5361124RP11-107P18 17p13.2 C-USP6 5415499 5602992RP11-27E24 17p13.2 C-USP6 5621843 5776426

2. Materials and methods

The Department of Pathology files were searched for caseswith the diagnoses of “giant cell reparative granuloma,”“aneurysmal bone cyst,” and “giant cell–rich lesions” between2000 and 2013. The criteria for selection included availabilityof nondecalcified tissue suitable for fluorescence in situhybridization (FISH) analysis. The clinical, radiographic, andmicroscopic findings were reviewed. The study was approvedby the institutional review board (no. WA0151-13 MSKCC).

2.1. Fluorescence in situ hybridization

FISH on interphase nuclei from paraffin-embedded 4-μmsections was performed applying custom probes usingbacterial artificial chromosomes (BAC), covering andflanking USP6 gene (Table 1). BAC clones were chosenaccording to UCSC genome browser (http://genome.ucsc.edu) and obtained from BACPAC sources of Children'sHospital of Oakland Research Institute (CHORI; Oakland,CA; http://bacpac.chori.org). DNA from individual BACswas isolated according to the manufacturer's instructions,labeled with different fluorochromes in a nick translationreaction, denatured, and hybridized to pretreated slides.Slides were then incubated, washed, and mounted withdiamidino-2-phenylindole in an antifade solution. Thegenomic location of each BAC set was verified byhybridizing them to normal metaphase chromosomes. Twohundred successive nuclei were examined using a Zeissfluorescence microscope (Zeiss Axioplan; Oberkochen,Germany), controlled by Isis 5 software (Metasystems,Newton, MA). A positive score was interpreted when atleast 20% of the nuclei showed a break-apart signal. Nucleiwith incomplete set of signals were omitted from the score.

3. Results

3.1. Clinical and radiographic features

Clinical features are summarized in Table 2. Forty-ninesamples from 48 patients (26 female and 22 male patients)were selected, including 17 lesions in the GCRG study group(9 from small bones of the hands and feet and 8 from gnathicsites) and 32 lesions in the control group (15 primary ABCfrom the long bones, 7 primary ABC from the flat bones,8 giant cell tumors [GCTs], and 2 brown tumors ofhyperparathyroidism).

The GCRG involving the hands and feet occurred in 9patients, 5 males and 4 females, with ages ranging from 9 to38 years (median, 16 years). Seven lesions involved thedigits, and 2 involved the calcaneus. On plain radiographs,all lesions had a lytic appearance, and all except 1 showedcortical destruction. Periosteal reaction was seen in 4 cases.Magnetic resonance imaging (MRI) was available in 8 of the

Table 2 Clinicopathological features and FISH results

Case no. Age Sex Site Diagnosis FISH for USP6

1 16 F Finger GCRG/solid ABC POS2 26 M Finger GCRG/solid ABC POS3 9 F Finger GCRG/solid ABC POS4 13 F Calcaneus GCRG/solid ABC POS5 16 M Calcaneus GCRG/solid ABC POS6 38 M Finger GCRG/ABC POS7 14 M Finger GCRG POS8 19 F Finger GCRG/ABC POS9 16 M Finger GCRG NEG10 36 F Mandible GCRG NEG11 54 F Mandible GCRG NEG12 5 M Maxilla GCRG NEG13 13 F Maxilla GCRG NEG14 70 M Maxilla GCRG NEG15 45 F Maxillary sinus GCRG NEG16 45 F Maxillary sinus GCRG NEG17 85 F Mandible GCRG NEG18 56 F Femur, distal Brown tumor NEG19 33 F Mandible Brown tumor NEG20 4 M Fibula ABC POS21 17 M Femur ABC NEG22 32 M Femur ABC NEG23 10 M Ulna ABC NEG24 15 M Humerus ABC NEG25 4 F Fibula ABC POS26 3 F Femur ABC NEG27 21 M Radius ABC POS28 9 F Tibia ABC POS29 15 F Tibia ABC NEG30 3 F Tibia ABC POS31 24 F Tibia ABC NEG32 29 F Ulna ABC POS33 22 M Fibula ABC NEG34 17 F Radius ABC POS35 27 F Orbit ABC POS36 18 M Pubis ABC POS37 14 F Manubrium ABC POS38 54 F Temporal bone ABC POS39 14 F Clavicle ABC POS40 18 F Clavicle ABC POS41 14 F Ilium ABC NEG42 33 M Tibia GCT with secondary ABC NEG43 16 M Tibia GCT with secondary ABC NEG44 49 M Patella GCT with secondary ABC NEG45 38 M Femur GCT with secondary ABC NEG46 43 M Femur GCT with secondary ABC NEG47 19 M Sacrum GCT with secondary ABC NEG48 36 M Finger GCT NEG49 51 F Finger GCT NEG

NOTE. Cases 1 to 9 represent lesions involving the bones of the hands and feet.Abbreviations: ABC, aneurysmal bone cyst; F, female; M, male; POS, positive; NEG, negative.

3USP6 gene rearrangements in giant cell reparative granuloma

9 cases, which showed a size range of 1.3 to 4.8 cm (mean,2.7 cm). The lesions were purely cystic in 2 cases, solid in 2cases, and mixed cystic and solid in 4 cases. Fluid-fluidlevels were seen in all except 2 cases, and extraosseousextension was seen in all except 1 case.

Gnathic GCRG included 8 lesions from 7 patients (5females and 2 males). The age at diagnosis ranged from 5 to85 years (median, 45 years). Five lesions from 4 patientsinvolved themaxilla, and 3 involved themandible. Computedtomographic findings were available in 6 of the 7 patients and

4 N. P. Agaram et al.

showed destructive enhancing lesions involving the bone,ranging from 1.3 to 4.5 cm, and extending into thesurrounding soft tissue. Of the 6 cases, 1 showed focal cysticchange. Of the 17 lesions in the GCRG study group, 15 wereprimary lesions, and 2 were recurrent lesions.

The ABCs of the long bones showed no sex predilectionand occurred in patients with an age distribution from 3 to 32years (median, 15 years), whereas those with ABCs of flatbones showed a 6:1 female/male ratio and an age range atdiagnosis of 14 to 54 years (median, 18 years). The sites ofinvolvement of the ABCs included tibia (4), femur (3),fibula (3), humerus (1), ulna (2), radius (2), clavicle (2), orbit(1), pubis (1), manubrium (1), temporal bone (1), and iliacbone (1). Radiographically, most ABCs were composed oflytic, expansile masses with well-defined borders. MRIhighlighted the characteristic internal septa and fluid-fluidlevels. The patients with GCT showed a male predilection,an age range at diagnosis of 16 to 51 years (median, 37years), occurring in the femur (2), tibia (2), finger (2), patella(1), and sacrum (1). Radiographically, GCT showed anexpansile growth, commonly involving the epimetaphysisand occasionally extending into surrounding soft tissues.The 2 brown tumors occurring in the setting of documentedhyperparathyroidism involved the mandible and the femur.Radiographically, both patients presented with multipleosseous lytic, expansile lesions involving the ribs and otherlong bones.

Fig. 1 GCRGof secondmetacarpal bone in a 16-year-old adolescent girllesion (arrows) with a Codman triangle (arrowhead) in a second metacarpallevels (arrowheads) in the expansile second metacarpal lesion mimicking atracer uptake (arrow) in the second metacarpal bone (R, right). D, Hematmagnification ×100), which, at higher power (E, ×200), reveals a mixture ogene break-apart assay showing an unbalanced rearrangement (arrows) wit

3.2. Pathologic findings

The GCRG occurring in the hands, feet, or gnathiclocation shared with the 2 brown tumors an osteoclast-typegiant cell–rich morphology associated with a spindle cellcomponent within a variable fibrous stroma. Areas ofhemorrhage and cyst formation were frequently noted. Fewlesions showed reactive new bone formation (Figs. 1 and 2).No cytologic atypia or increased mitotic activity was noted.

The ABC lesions of the long and flat bones showeddistinctive cystic hemorrhagic areas, with fibrous cyst wallscomposed of spindle to histiocytoid cells admixed withosteoclast-like giant cells and variable osteoid matrixdeposition. Few lesions showed focally more solid areas,with morphology indistinguishable from the GCRGs.

The GCTs showed a uniform distribution of giant cells ina background of mononuclear cells. Areas of hemorrhagewere frequently noted. Of the 8 cases, 6 showed presence of asecondary ABC component.

3.3. FISH findings

FISH forUSP6 was performed on all 49 cases in the study(Table 2). Of the 9 lesions of the hand and feet, 8 (89%)showed rearrangements of the USP6 gene (Figs. 1 and 2). Incontrast, none of the 8 gnathic GCRGs showed USP6 gene

(case 1). A, The anterior-posterior radiograph shows an expansile lyticbone. B, The MRI axial STIR image of the hand shows multiple fluidn ABC. C, The spot view of the bone scan shows intense radioactiveoxylin and eosin sections showing a giant cell–rich lesion (originalf bland spindle cells and osteoclast-like giant cells. F, FISH forUSP6h deletion of the telomeric (green) signal (red, centromeric signal).

Fig. 2 GCRG in the fourth proximal phalanx of a 38-year-old man (case 6). A, Anterior-posterior radiograph of the fourth finger shows aneccentric lesion (arrow) in the proximal phalanx. B, MRI axial STIR image shows the lesion (arrows) eroding the bone. C, MRI axial T1-weighted image shows an enhancing lesion (arrowhead) eroding the bone. D, Hematoxylin and eosin section (×200) reveals lesional spindlecells with admixed osteoclast-like giant cells. E, FISH showing USP6 break-apart signal (arrows) with associated deletion of the telomeric(green) signal (red, centromeric signal).

5USP6 gene rearrangements in giant cell reparative granuloma

abnormalities. From the control group, USP6 gene rearran-gements were identified in 13 (59%) of 22 ABCs from thelong and flat bones. No gene abnormalities were identified inany of the brown tumors or the GCT of bone with or withoutsecondary ABC changes. The pattern of USP6 geneabnormalities was similar in the lesions of the hands andfeet and the primary ABCs from long and flat bones with thegenetic abnormalities seen only in the spindle cells and not inthe osteoclast-like giant cells.

4. Discussion

The recent identification of recurrent chromosomaltranslocations involving USP6 gene in primary ABC haschanged our understanding of this disease, from a reactive,nonneoplastic process, possibly initiated by injury to thecapillary network leading to an expansile destructiveprocess, to a clonal, truly neoplastic lesion. The solid variantof ABC has been shown to harbor similar genetic events asthe classic cystic lesions.

USP6 is a ubiquitin-specific protease, the gene of whichhas been localized to the short arm of chromosome 17(17p13). The gene has been postulated to contribute tohominoid speciation [14]. The oncogenic mechanism bywhich USP6-related gene fusion is involved in tumorigen-esis, as studied in ABCs, is by promoter swapping whereinthe juxtaposition of USP6 gene to a highly active CDH11promoter leads to USP6 up-regulation [11]. Apart from thebone lesions such as ABCs, other lesions wherein USP6 hasbeen implicated in the pathogenesis include nodular fasciitisand a subset of myositis ossificans [15,16]. Interestingly,both of these tumors have for long been regarded as reactivelesions in the soft tissue.

The terminology related to the giant cell–rich lesions ofthe hands and feet has been long controversial, withdesignations including both GCRG and solid ABC, due totheir signficant morphologic overlap. Given that ABC is aclonal process and GCRGs have always been considered areactive, nonneoplastic process involving gnathic andextragnathic locations, the combined terminology ofGCRG and/or solid ABC is ambiguous and does not

6 N. P. Agaram et al.

shed light on the true biologic nature of the lesion.Furthermore, the morphologic similarities between gnathicand extragnathic GCRGs raise the question if lesionsoccuring at different sites share similar genetic alterationsand biologic potential. In an attempt to address theseissues, we investigated a series of lesions of the gnathicGCRGs and giant cell–rich lesions of hands and feet incomparison with primary ABCs and GCTs of long bones.

None of the 8 gnathic GCRGs in our study showed USP6abnormalities. In contrast, 8 of the 9 lesions from hands andfeet showed rearrangements of the USP6 gene. These resultsindicate that the 2 groups have different genetic abnormal-ities and that the so-called GCRGs of the small bones ofhands and feet are true neoplastic processes, in keeping withABC. Thus, we recommend that the terminology of GCRGof the hands and feet should be abandoned and replacedinstead with either ABC or solid variant of ABC dependingon the radiologic findings.

Our study also confirms that gnathic lesions arepathogenetically different from those occuring in extra-gnathic locations, although they share significant morpho-logic overlap. The terminology of GCRG should be reservedmainly for lesions occuring in the gnathic location.

USP6 rearrangements were identified in 59% of the ABCsin our study. This is in keeping with the reported rates ofUSP6rearrangement (69%) as reported in the literature [12]. None ofthe GCT of bone showed USP6 rearrangements.

Brown tumor of hyperparathyroidism is a bone lesionoccuring in the setting of primary or secondary hyperpara-thyroidism. Radiographically, these are lytic lesions. Histo-logically, they show giant cell–rich areas with associatedfibrosis and new bone formation and are indistinguishableform other giant cell–rich lesions such as solid ABC, GCRG,or GCT of bone. Clinical history along with serum calcium,phosphorous, and parathormone levels helps in diagnosis ofthese lesions. None of the 2 cases in our study showed USP6gene abnormalities, findings in keeping with the study bySukov et al [16] who showed absence of USP6 abnormalitiesin 6 cases of brown tumors.

In summary, most of the so-called GCRG of the handsand feet are truly solid ABCs and should be classified assuch. In our opinion, the terminology of GCRG should berestricted to the gnathic lesions with this morphology andshould be avoided in extragnathic sites. Our study alsovalidates the usefulness of FISH analysis for USP6 generearrangements as an ancillary tool to separate ABCs fromother giant cell–rich lesions such as GCRG and GCT ofbone, especially in the setting of lesions in small bones whereit may difficult to distinguish these entities based on clinical,radiologic, and morphologic findings.

Acknowledgments

The authors would like to thank Lorraine Biedrzycki forpreparation of composite figures and Milagros Soto foreditorial assistance.

References

[1] Jaffe HL. Giant-cell reparative granuloma, traumatic bone cyst, andfibrous (fibro-osseous) dysplasia of the jawbones. Oral Surg Oral MedOral Pathol 1953;6:159-75.

[2] Ackerman LV, Spjut HJ. Tumors of bone and cartilage. Washington,D.C.: Armed Forces Institute of Pathology; 1962.

[3] Lorenzo JC, Dorfman HD. Giant-cell reparative granuloma of shorttubular bones of the hands and feet. Am J Surg Pathol 1980;4:551-63.

[4] Sanerkin NG, Mott MG, Roylance J. An unusual intraosseous lesionwith fibroblastic, osteoclastic, osteoblastic, aneurysmal and fibromyx-oid elements. “Solid” variant of aneurysmal bone cyst. Cancer1983;51:2278-86.

[5] Ratner V, Dorfman HD. Giant-cell reparative granuloma of the handand foot bones. Clin Orthop Relat Res 1990:251-8.

[6] Bertoni F, Bacchini P, Capanna R, et al. Solid variant of aneurysmalbone cyst. Cancer 1993;71:729-34.

[7] Oda Y, Tsuneyoshi M, Shinohara N. “Solid” variant of aneurysmalbone cyst (extragnathic giant cell reparative granuloma) in the axialskeleton and long bones. A study of its morphologic spectrum anddistinction from allied giant cell lesions. Cancer 1992;70:2642-9.

[8] Ilaslan H, Sundaram M, Unni KK. Solid variant of aneurysmal bonecysts in long tubular bones: giant cell reparative granuloma. AJR Am JRoentgenol 2003;180:1681-7.

[9] Panoutsakopoulos G, Pandis N, Kyriazoglou I, Gustafson P, MertensF, Mandahl N. Recurrent t(16;17)(q22;p13) in aneurysmal bone cysts.Genes Chromosomes Cancer 1999;26:265-6.

[10] Dal Cin P, Kozakewich HP, Goumnerova L, Mankin HJ, RosenbergAE, Fletcher JA. Variant translocations involving 16q22 and 17p13 insolid variant and extraosseous forms of aneurysmal bone cyst. GenesChromosomes Cancer 2000;28:233-4.

[11] Oliveira AM, Hsi BL, Weremowicz S, et al. USP6 (Tre2) fusiononcogenes in aneurysmal bone cyst. Cancer Res 2004;64:1920-3.

[12] Oliveira AM, Perez-Atayde AR, Inwards CY, et al. USP6 and CDH11oncogenes identify the neoplastic cell in primary aneurysmal bonecysts and are absent in so-called secondary aneurysmal bone cysts. AmJ Pathol 2004;165:1773-80.

[13] Oliveira AM, Perez-Atayde AR, Dal Cin P, et al. Aneurysmal bonecyst variant translocations upregulate USP6 transcription by promoterswapping with the ZNF9, COL1A1, TRAP150, and OMD genes.Oncogene 2005;24:3419-26.

[14] Paulding CA, Ruvolo M, Haber DA. The Tre2 (USP6) oncogene is ahominoid-specific gene. Proc Natl Acad Sci U S A 2003;100:2507-11.

[15] Erickson-Johnson MR, Chou MM, Evers BR, et al. Nodular fasciitis: anovel model of transient neoplasia induced by MYH9-USP6 genefusion. Lab Invest 2011;91:1427-33.

[16] Sukov WR, Franco MF, Erickson-Johnson M, et al. Frequency ofUSP6 rearrangements in myositis ossificans, brown tumor, andcherubism: molecular cytogenetic evidence that a subset of “myositisossificans-like lesions” are the early phases in the formation of soft-tissue aneurysmal bone cyst. Skeletal Radiol 2008;37:321-7.