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Aneurysmal bone cyst is a benign, often rapidly expanding osteolytic multicystic lesion that was first described by Jaffe and Lichtenstein1 in 1942. Aneurysmal bone cyst most commonly arises in young patients and typically involves the metaphyseal region of long bones and vertebrae.2, 3 Histopathologically, the lesion is composed of varying sized blood-filled spaces separated by connective tissue septa containing bony trabeculae and giant osteoclastic cells.4 Aneurysmal bone cysts are frequently locally aggressive and exhibit a strong tendency to recur.5

Relatively few cases of aneurysmal bone cyst have been cytogenetically characterized.3, 6, 7, 8, 9, 10, 11, 12, 13 Clonal abnormalities of the short arm of chromosome 17 appear to be recurrent and some authors have postulated that alterations of a ‘yet to be determined’ gene on 17p13 may play an important role in the development of aneurysmal bone cyst.3, 7, 8, 9, 10, 12, 13 In this study, the cytogenetic findings of 43 aneurysmal bone cyst samples from 38 patients are reported; several exhibiting novel rearrangements. Moreover, molecular cytogenetic data collected from efforts to further localize the key breakpoint on 17p are described.

Materials and methods

Clinical

Over a 12-year period, representative tissue samples of 43 aneurysmal bone cyst specimens from 38 patients were received for cytogenetic analysis within 36 h of surgery. The clinicopathologic data of all patients are summarized in Table 1. A total of 22 male and 16 female patients with ages ranging from 4 to 48 years were analyzed. In all, 41 of the 43 specimens studied were primary lesions and two were recurrent. Both the biopsy and definitive surgical specimen were available for cytogenetic analysis for four patients. All specimens analyzed were intraosseous and arose de novo with the exception of case 35 that was a secondary aneurysmal bone cyst (associated with a chondroblastoma; also reviewed by Dr K Unni, Mayo Clinic). Excluding case 35, all cases exhibited typical radiographic and microscopic features of aneurysmal bone cyst; that is, lytic, eccentric, expansile masses with well-defined margins histologically composed of blood-filled cystic spaces separated by loose fibroconnective tissue with prominent giant cell reaction and focal reactive bone formation. No solid variant aneurysmal bone cysts were seen.

Table 1 Clinicopathologic, cytogenetic and molecular cytogenetic data
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Cytogenetic Analysis

Cytogenetic analysis was performed on sterile, representative tissue of each case utilizing standard culture and harvest procedures as described previously.14, 15 Briefly, the tissues were disaggregated mechanically and enzymatically and cultured in RPMI 1640 supplemented with 20% fetal bovine serum for 3–8 days. Cells were exposed to an overnight treatment of Colcemid (0.02 μg/ml). Following hypotonic treatment (0.8% sodium citrate for 20 min), the preparations were fixed three times with methanol:glacial acetic acid (3:1). Metaphase cells were banded with Giemsa trypsin. The karyotypes were expressed according to the International System for Human Cytogenetic Nomenclature.16

Probe Design and Development

Based on findings from a previous study12 suggesting that the putative 17p13 gene involved in aneurysmal bone cyst was located within the region between the TP53 (17p13.1) and Miller–Dieker lissencephaly syndrome (MDS, 17p13.3) genes, FISH studies were initiated to further define the critically involved breakpoint. This was accomplished by dividing the roughly 5.0 Mb region of interest into 16 segments that sequentially spanned the entire region (Figure 1). Bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) probes for each section were identified utilizing the National Center for Biotechnology (NCBI)17 and the Wellcome Trust Sanger Institute Project Ensembl18 databases. Combinations of probe sets were fashioned to flank each defined subregion.

Figure 1
figure 1

This schematic illustrates the different probe set combinations used to sequentially examine 15 subdivided regions spanning a 5.1 Mb sector flanked by the TP53 (17p13.1) and the MDS (17p13.3) gene loci. The green boxes indicate probe sets labeled in Spectrum Green and the red boxes indicate probe sets labeled in Spectrum Orange. A Spectrum Aqua α-satellite probe for the centromeric region of chromosome 17 was employed for ploidy determination.

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Molecular Cytogenetic Analysis (FISH)

Tricolor FISH studies were performed on clonally abnormal metaphase cells [exhibiting the t(16;17) (q22;p13)] and/or cytologic touch preparations of lesional tissue from 35 specimens using BAC and PAC probes (Children's Hospital Oakland Research Institute, Oakland, CA, USA and CTA-D library, Research Genetics, Huntsville, AL, USA) localized to 17p13.1–p13.3 (Figure 1). Probes were nick translated with either Spectrum Green or Spectrum Orange-dUTP utilizing a modification of the manufacturer's protocol (Vysis, Downers Grove, IL, USA). An amount of 1 μg of DNA for each of two or three probes were combined. All nick translation reagents were then multiplied by the total μg of DNA used in the cocktail. Amounts of 200 ng of each probe were hybridized and blocked approximately 15 times with a combination of Human Cot-I DNA (Invitrogen, Carlsbad, CA, USA) and human placental DNA. A spectrum aqua α-satellite probe for the centromeric region of chromosome 17 (Vysis) was employed for ploidy determination.

Prior to hybridization, slides were pretreated at 72°C in 2 × SSC for 2 min and at 37°C in pepsin working solution (20 μl 10% pepsin in 50 ml of 0.1 N HCl) for 3 min. Following pretreatment, the cells and probes were codenaturated at 75°C for 1 min and incubated overnight at 37°C using the HYBrite™ system (Vysis, Downers Grove, IL, USA). Hybridization signals were assessed in 10 metaphase cells or in 200 interphase nuclei with strong, well-delineated signals by two different individuals. An interphase cell specimen was interpreted as abnormal for the 17p13.2 locus if a split of flanking probe signals or loss of the distal flanking probe signal was detected in >10% of the cells evaluated (more than two standard deviations above the average false positive rate). As additional controls, normal peripheral blood lymphocytes, cytologic touch preparations of normal skeletal muscle, and cytologic touch preparations of a unicameral bone cyst were simultaneously hybridized with the same probe sets. Images were acquired using the Cytovision Image Analysis System (Applied Imaging, Santa Clara, CA, USA).

Results

Conventional Cytogenetic Findings

Metaphase cells were obtained in all 43 specimens examined with clonal chromosomal abnormalities detected in 12 (Table 1). Eight specimens exhibited karyotypic anomalies of 17p (cases 15A, 15B, 28, 29, 31, 32, 34, and 37). A balanced 16;17 translocation [t(16;17)(q22;p13)] was observed in three specimens (cases 15A, 15B, and 28, Figure 2). An additional specimen (case 34) showed the 16;17 translocation in the form of a three-way translocation [t(7;17;16)(q21;p13;q22)]. Four additional cases demonstrated 17p13 rearrangements with chromosomal partners other than chromosome 16 (cases 29, 31, 32, and 37, Figure 3). Reciprocal translocations involving chromosomes 1 and 7 were detected in two cases (cases 13 and 24), albeit involving different breakpoints. Moreover, the 1;7 translocation observed in case 24 was shown to be a constitutional anomaly following chromosomal analysis of a peripheral blood sample of this patient.

Figure 2
figure 2

(a) Representative schematic and GTG-banded partial karyotype of the 16;17 translocation [t(16;17)(q22;p13.2)] observed in case 28. Representative GTG-banded (b) and FISH (c) metaphase cell images of case 28 confirm a split of one of the ‘region seven’ flanking probe sets with translocation of the distal probe set signal (orange) to the der (16) of the t(16;17)(q22;p13). The aqua signals indicate the centromeric regions of the normal and derivative chromosome 17 homologues and the green signals the proximal probe portion of the ‘region seven’ flanking probe set.

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Figure 3
figure 3

(a) Partial GTG-banded karyotype of case 29 illustrating the 6;17 translocation [t(6;17)(p21.2;p13)]. (b) FISH analysis performed on cytologic touch preparations of case 29 showing a representative interphase cell with disruption of ‘region seven’ (single green and single orange signals) and one normal chromosome 17 homologue (juxtaposed orange and green signals). The aqua signals represent the centromeric region of the chromosome 17 homologues. (c) Partial GTG-banded karyotype of case 37 illustrating an inversion of one chromosome 17 homologue (upper and lower arrows indicate breakpoints 17p13 and 17q11.2–12, respectively). (d) FISH analysis performed on cytologic touch preparations of case 37 showing a representative interphase cell with disruption of ‘region seven’ (single green and single orange signals) and one normal chromosome 17 homologue (juxtaposed orange and green signals). The aqua signals represent the centromeric region of the chromosome 17 homologues. (e) Representative GTG-banded karyotype of case 38 showing normal results. (f) FISH analysis performed on cytologic touch preparations of case 38 showing a representative interphase cell with loss of the distal portion of the ‘region seven’ flanking probe set (single green signal without accompanying orange signal) and one normal chromosome 17 (juxtaposed orange and green signals). The aqua signals represent the centromeric region of the chromosome 17 homologues.

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Molecular Cytogenetic Findings

Initial FISH studies were performed on cytologic touch preparations of lesional tissue from case 15B characterized by a cytogenetically confirmed t(16;17)(q22;p13) with 15 separate flanking probe combinations covering all 16 segments from the 5.1 Mb region (Figure 1). Only ‘region seven’ (Spectrum Orange-dUTP labeled proximal probes: RP11-46I8, RP11-333E1, RP11-457I8; Spectrum Green-dUTP labeled distal probes: RP11-198F11, RP11-115H24, RP5-1050D4) showed a split of the proximal and distal probe sets indicating a disruption of this 17p13.2 locus. Subsequent FISH studies performed on t(16;17) metaphase cells from case 28 with the ‘region seven’ cocktail probes confirmed signal splitting of this probe set with translocation of the distal probe set to the der(16), (Figure 2). Lastly, the 33 additional aneurysmal bone cyst specimens subjected to FISH with the ‘region seven’ probe set showed the following results: (1) In all, 13 specimens (cases 2, 5, 12B, 16A, 20, 22, 29, 31, 32, 33, 34, 36, and 37) showed a split of one set of probe signals (Figure 3); (2) six specimens (cases 9, 17, 23, 24, 26, and 38) showed loss of the probe set signal distal to ‘region seven’ of the 17p13.2 locus (Figure 3), and; (3) one case demonstrated an extra copy of the probe set signal proximal to ‘region seven’ of the 17p13.2 locus (case 18).

Discussion

Previous cytogenetic studies of aneurysmal bone cyst have revealed 17 cases with clonal chromosomal abnormalities (Table 2),3, 6, 7, 8, 9, 10, 11, 12, 13 Six of these 17 (35%) cases showed a t(16;17)(q22;p13), while seven additional cases demonstrated rearrangement of 17p11–13 with a chromosome partner other than 16q22. Conversely, 16q22 abnormalities exclusive of 17p11–13 abnormalities have been detected in two aneurysmal bone cysts.7, 10 In the current study, three specimens from two patients exhibited an identical t(16;17)(q22;p13) whereas one specimen demonstrated a novel, complex variant translocation, t(7;17;16)(q21;p13;q22). Other anomalies of 17p13 were observed in four additional specimens. Overall, the following chromosomal bands have been detected as 17p11–13 rearrangement partners in aneurysmal bone cyst: 1p34.1–34.3, 2p23, 6p21, 9p22, 14q11.2, 16q22, and 17q12.3, 6, 7, 8, 9, 10, 11, 12, 13 Observation of repeated involvement of 17p11–13 suggests that a putative oncogene important in the pathogenesis of aneurysmal bone cyst lies within this chromosomal region and the manner of its activation is diverse based on the variable 17p chromosomal rearrangement partners.19

Table 2 Chromosome changes in aneurysmal bone cyst
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In addition to the apparent nonrandom partnership of 16q22 and 17p11–13, the current study reveals two more breakpoints that appear to be recurrently rearranged with 17p13. Winnepenninckx et al12 reported a case of aneurysmal bone cyst in the nose of a 6-year-old girl that exhibited a t(6;17)(p21;p13). Similarly, case 29, an aneurysmal bone cyst arising in the distal fibula of a 13-year-old female, showed an identical 6;17 translocation (Figure 3). Rearrangements of 6p have been seen in several types of tumors of mesenchymal, epithelial, and hematopoietic origin among which are lipoma, uterine leiomyoma, hamartoma, pediatric renal epithelial neoplasm, ovarian carcinoma, and follicular lymphoma.20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 The putative oncogene, HMGIY (high mobility group protein isoforms I and Y), localized to 6p21 has been proposed to play a role in the development of a number of these neoplasms.20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34

An identical t(17;17)(q12;p13) was seen in two previous studies.7, 9 Interestingly, an inversion of chromosome 17 [inv(17)(p13q11.2–12)] involving similar breakpoints was observed in case 37 of the present study (Figure 3). Although rearrangements of the 17q12 band have not been reported as prominent in mesenchymal tumors, the 17q12 locus has received considerable attention because of the localization of the Her-2neu (a.k.a. c-erbB-2, erythroblastic leukemia viral oncogene homolog 2) and RARA (retinoic acid receptor, alpha) genes to this chromosomal band. Assessment of the copy number of the Her-2neu gene and its expression has evolved as an important prognostic indicator in breast, ovarian, and uterine cancers.35, 36, 37, 38, 39 The characteristic rearrangement of RARA with the PML (promyelocytic leukemia) gene on 15q22 has proven to be integral in the diagnosis and treatment of acute promyelocytic leukemia.40, 41, 42

Since the cytogenetic findings of the current study and a review of the literature indicated that the 17p11–13 region likely harbors an oncogene of etiologic importance in aneurysmal bone cyst, efforts to further characterize the critically involved 17p breakpoint were conducted. FISH studies performed on 35 specimens covering a 5.1 Mb region flanked by the TP53 (17p13.1) and MDS (17p13.3) gene loci revealed the key breakpoint lies within a 250 kb region located approximately 5.2–5.5 Mb from the telomere of 17p and bound by RP11-46I8 and RP5-1050D4 at 17p13.2. Approximately 12 genes have been mapped to this 250 kb area.17, 18, 43, 44 Perhaps more specifically, the authors speculate that the exact breakpoint lies within a 15–30 kb region of junctional overlap between BAC probe RP11-46I8 and PAC probe RP5-1050D4, although the relatively gross nature of the approach precludes precise characterization. One gene, FLJ30726, is localized within this smallest region of overlap.44 FLJ30726 is a temporary designation for this gene that encodes for a protein with an unknown function but bears moderate similarity to the zinc-finger protein 35 (ZNF35).44 The ZNF35 protein is believed to be a sequence-specific nucleic acid-binding protein that may function as a transcriptional activator.45, 46

In conclusion, cytogenetic abnormalities of the short arm of chromosome 17 are prominent in aneurysmal bone cyst. Unfortunately, conventional cytogenetic approaches have revealed clonal karyotypic abnormalities in only a fraction of cases examined (12/43 (28%) specimens in the present study), thereby limiting the prospective usefulness of this approach as an adjunct in diagnostically vexing cases. On the other hand, the results of the current study show that FISH probe sets developed to further localize the critical 17p breakpoint, allow detection of 17p13.2 aberrations in a greater subset of aneurysmal bone cysts (22 of 35 (63%) specimens), thereby enhancing the potential diagnostic utility of this assay. Moreover, this FISH approach can also be performed on nondividing cells of aneurysmal bone cysts. Lastly, the more precise localization of the critical 17p breakpoint in this study should provide direction for future molecular studies aimed at deciphering the underlying gene that may prove central to aneurysmal bone cyst development.