JMD 2001, Vol. 3, No. 1
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology
Extra Copies of Chromosomes 7, 8, 12, 19, and 21 are Recurrent in Adamantinoma
Masahiko Kanamori*,
Cristina R. Antonescu§,
Melody Scott*,
Robert S. Bridge, Jr.*,
James R. Neff
,
Suzanne S. Spanier¶,
Mark T. Scarborough||,
Gerardo Vergara**,
Howard G. Rosenthal** and
Julia A. Bridge*,
From the Departments of Pathology and Microbiology,
*
Orthopaedic Surgery,
and Pediatrics,
Center for Human Molecular Genetics, University of Nebraska Medical Center, Omaha, Nebraska; the Department of Pathology,
§
Memorial Sloan-Kettering Cancer Center, New York, New York; the Departments of Pathology
¶
and Orthopaedic Surgery,
||
Shards Hospital, Gainesville, Florida; and Trinity Lutheran Hospital,
**
Kansas City, Missouri
 |
Abstract
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Adamantinoma of long bones is a rare neoplasm predominantly
involving the tibia. Cytogenetic studies of adamantinoma are few.
Cytogenetic or molecular cytogenetic analysis of four
adamantinomas, and a review of eleven cases in the
literature reveals extra copies of chromosomes 7,
8, 12, 19, and 21 as recurrent in this
neoplasm. Adamantinoma may be confused with a variety of primary and
metastatic epithelial and mesenchymal neoplasms. Observation of these
aneuploidies may be useful in establishing the diagnosis of
adamantinoma.
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Introduction
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Adamantinoma of long bones is a rare low-grade malignant bone
tumor most often arising in the tibia, but also occasionally exhibiting
synchronous involvement of the ipsilateral fibula. Some adamantinomas
have "osteofibrous dysplasia-like" features.1
Thus, a
relationship between adamantinoma and osteofibrous dysplasia (a benign
fibro-osseous lesion occurring almost exclusively in the tibia of
children less than 10 years of age) has been proposed. This
relationship, however, is poorly understood.
Cytogenetic analyses of adamantinoma and osteofibrous dysplasia are
few.2, 3, 4, 5, 6, 7
In this study, the cytogenetic and molecular
cytogenetic findings of four cases of adamantinoma and a review of the
literature are presented.
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Materials and Methods
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Case 1
A 37-year-old female presented with a nontender protuberance in
the midshaft of the right tibia known since childhood. As a child, she
sustained a fracture of the tibia with minimal trauma. The fracture was
treated in a cast and healed without complications. For 20 years, the
protuberance remained unchanged in size and character and without
symptoms. In 1999, however, she sustained a fracture of the tibia once
again and with minimal trauma. Plain radiographs demonstrated a
pathological fracture through a destructive process including the
lesion and protuberance. A biopsy revealed adamantinoma. A radical
en bloc resection of the right tibial diaphysis and
reconstruction using fresh frozen allograft tibia, plates, and
autologous bone graft were performed.
Histopathologically, both the biopsy and resected specimens were
composed of epithelial-like cells arranged in large islands or nests
embedded in a densely fibrous stroma. Mitotic figures could not be
identified. Immunohistochemical staining revealed that the epithelial
cells were immunoreactive for cytokeratin (AE1:AE3). The
histopathological diagnosis was classic adamantinoma.
Case 2
A 20-year-old male sought medical attention because of pain in the
anterior aspect of his right leg with recreational running. Four years
earlier, the patient had noted a bulge on the anterior border of his
right tibia. Plain radiographs revealed a bubbly lesion involving the
anterolateral cortex of the midshaft of the right tibia. Comparison
with previous radiographs over the past 4 years showed indolent
progression (Figure 1)
. Magnetic resonance imaging (MRI) demonstrated a multilobulated
mass that uniformly enhanced with contrast. A bone scan showed intense
uptake confined to the site of the lesion seen in the plain radiographs
and revealed no other areas of significant skeletal uptake. An
incisional biopsy was followed by a wide local excision and
reconstruction.
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Figure 1. The Case 2 lesion involving the anterior cortex has multiple
radiolucent defects surrounded by areas of cortical thickening and a
thick rim of reactive bone at the base. One can observe indolent
progression of the radiolucent areas from the first film taken July,
1995 (left) to
subsequent ones obtained May, 1996
(center) and
December, 1999
(right).
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Microscopically, the lesional tissue was intracortical. At the
periphery, the cortex demonstrated enlarged Harversian canals filled
with a paucicellular fibroblastic reactive tissue. There was a
transition from mature to less mature bone tissue, or so-called zoning.
The center of the lesion consisted of a loosely textured fibroblastic
proliferation with occasional rounded or oval deposits of nonlamellar
bone without noticeable osteoblastic rimming and nests of
epithelial-like cells (Figure 2)
. Immunohistochemical staining for cytokeratin (AE1:AE3) was positive
(Figure 2)
. The histopathological diagnosis was differentiated
adamantinoma.
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Figure 2. A: The medullary side of a portion of the reactive cortex is
at the top of the left hand corner. It is fairly mature, but lacks
well-developed Harversian systems and stress lines. Connected to it and
protruding toward the underlying lesion, are thickened trabeculae of
irregular, immature bone variably rimmed by osteoblasts. Osteoclastic
remodeling can be seen in the lower right hand corner. B:
The center of the lesion is characterized by a bland proliferation of
cells having spindle shaped, often slender, nuclei arranged in a loose
storiform pattern. Embedded within this stroma are two nests of
epithelial cells. C: An immunohistochemical preparation for
cytokeratins highlights three nests of epithelial cells. In addition,
multiple keratin reactive single cells within the stroma, not otherwise
detectable, can be appreciated.
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Case 3
A 16-year-old female was medically evaluated for an enlarging,
painful lesion in the midshaft of the right tibia. The MRI demonstrated
a lesion occupying the marrow of the diaphysis of the tibial shaft
containing multiple fluid-fluid levels. Extensive endosteal erosion,
cortical thinning, and a small associated extraosseous soft tissue mass
were noted. The ipsilateral fibula was unremarkable. An open biopsy was
performed resulting in a tissue diagnosis of adamantinoma. The patient
underwent a wide local excision of the midshaft tibia using the
contralateral revascularized osteocutaneous fibula as a graft for
reconstruction. The resected specimen showed two separate hemorrhagic
and partly cystic lesions in the mid and distal tibial shaft, measuring
7.0 and 2.4 cm, respectively, with cortical breakthrough into the
adjacent soft tissues. Microscopically both foci had similar features,
large nests of epithelioid cells in a basaloid pattern, characteristic
of classic adamantinoma. The tumor cells were strongly immunoreactive
for 34BE12 and BCL2 and focally immunoreactive for cytokeratin
(AE1:AE3).
Case 4
A 14-year-old male was evaluated for increasing pain and swelling
of his right leg. His past medical history revealed a long record of
lesions involving his right tibia and fibula since the age of 2,
including three surgical procedures performed on these lesions at ages
4, 7, and 11. A curettage without graft of the tibia was performed for
a pathological fracture at age 4, diagnosed at an outside hospital as a
unicameral bone cyst. At age 8, the patient underwent a resection of a
fibular lesion, which was diagnosed as osteofibrous dysplasia. At age
11, he had a curettage and grafting of a tibial lesion, which also
demonstrated features consistent with osteofibrous dysplasia. Plain
films revealed a lytic lesion in the distal tibia, with significant
endosteal scalloping. The entire posterior lateral cortex was
destroyed, and on MRI a large soft tissue component projecting into the
posterior compartment was noted.
Lesional tissue obtained from an initial curettage of the right
tibia was diagnostic of classic adamantinoma. The tumor cells were
positive for cytokeratins (CAM 5.2, AE1:AE3, and 34BE12). The patient
underwent a wide below-knee amputation procedure. The resected specimen
showed an 8-cm mass primarily in the soft tissues of the posterior
compartment of the right calf, with focal involvement of the posterior
cortex of the tibia. The histopathological appearance was similar to
that of the curettage specimen. The mitotic count was 20MF/10HPF. No
skip lesions or osteofibrous-dysplasia-like changes were identified.
Cytogenetic Analysis
Cytogenetic analyses were performed on representative, sterile
tissue removed from the resected specimens of Cases 1 and 2. Standard
culture and harvesting procedures were used which have been described
previously.5
Briefly, the tissue was disaggregated
mechanically and enzymatically and cultured in RPMI 1640 media
supplemented with 20% fetal bovine serum, 1%
penicillin/streptomycin-L-glutamine (Irvine Scientific, Santa Ana, CA)
for 4 to 7 days. Four hours before harvest, cells were exposed to
Colcemid (0.02 mg/ml). After hypotonic treatment (0.074 mol/L
KCI for 30 minutes), the preparations were fixed 3 times with methanol
and 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 1995.8
Molecular Cytogenetic Analysis
Fluorescence in situ hybridization (FISH) studies were
performed on cytologic touch preparations of Cases 3 and 4 using
chromosomes 7-, 8-, 12-, and 19-specific probes (CEP7, Vysis, Inc.,
Downers Grove, IL; D8Z1 and D12Z1, Oncor, Inc., Gaithersburg, MD;
chromosome 19q12–13.1 genomic PAC probe, F1, courtesy of M. Zielenska
and J.A. Squire, Toronto9
).
Hybridization was performed as previously described.6
The
number of hybridization signals for each specimen was assessed in 200
interphase nuclei with strong and well-delineated signals by two
different individuals. A specimen was interpreted as aneuploid for
chromosomes 7, 8, 12, and/or 19 if three or more clearly separate
signals for each respective probe could be detected in >25% of the
cells evaluated (more than two standard deviations above the average
false positive rate). To exclude the possibility of triploidy or
tetraploidy, the biotinylated probe, D3Z1 (Oncor, Inc.), which binds to
the centromeric region of chromosome 3, was used. As an additional
control, normal peripheral blood lymphocytes were simultaneously
hybridized with chromosomes 3, 7, 8, 12, and 19 probes. Images were
prepared using the Applied Image Analysis System (Applied Imaging,
Pittsburgh, PA).
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Results
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Cytogenetics
Six metaphase cells were analyzable from Case 1. The following
abnormal chromosomal complement was detected in five cells:
54,XX,-1,+5,+der(7)t(?1;7)(?q21;q22),
+der(8)t(1;8)(q21;q24.3), +der(9)t(1;9)(p32;q34), +19,
+20,+21,+mar1,+mar2 (Figure 3)
. One cell was karyotypically normal.
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Figure 3. GTG-banded karyotype of Case 1: 54,
XX,-1,+5,+der(7)t(?1;7)(?q21;q22),+der(8)t(1;8)(q21;q24.3),+der(q)t(1:9)(p32;q34),+19,+20,+ 21,+mar1,+mar2.
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The following primary abnormal clone and subclone were detected
in Case 2: 54,XY,+5,+7,+8,+12,+12,+14, +19, +21[6]/53, XY,
+4, +5, +7, +8, +12, +14, -16, +17, -18,+21,+mar[2]. A
representative karyotype of the primary clone is presented in Figure 4
. Six cells were karyotypically normal.
Molecular Cytogenetics
Trisomy for chromosomes 7, 8, and 19 were detected in 63 to 73%
of the cells examined in Case 3. Two copies (diploid copy number) of
chromosomes 3 and 12 were detected in 97% and 96% of the cells,
respectively, for Case 3.
Assessment of the control probe (chromosome 3) revealed that the
majority of cells (83%) in Case 4 were tetraploid. The copy number of
chromosome 8 probe signals was five or more in 84% of the 200 cells
evaluated and was thus interpreted as abnormal in this case. Four
signals (tetraploid copy number) for chromosomes 7, 12, and 19 probes
were seen within the control range for this case. Representative
interphase cells from both Cases 3 and 4 after FISH analyses are
illustrated in Figure 5
.
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Figure 5. A: FISH analysis performed on cytologic touch preparations
of Case 3 revealed three hybridization signals for chromosome 8
(red) and two
hybridization signals for chromosome 12
(green). B:
FISH analysis performed on cytologic touch preparations of Case 3
revealed three hybridization signals for chromosome 19. C:
FISH analysis performed on cytologic touch preparations of Case 4
revealed five to eight hybridization signals for chromosome 8
(red) and four
hybridization signals for chromosome 12
(green).
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Discussion
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Adamantinoma of the appendicular skeleton is a distinct,
exceptionally rare neoplasm of controversial histopathogenesis. The
first report of an adamantinoma of long bone ("primary myelogenic
squamous cell carcinoma of the ulna") was described by Maier in
1900.10
This description of a diaphyseal ulnar lesion
corresponds to the currently accepted diagnostic criteria for
adamantinoma. Only 195 well-documented cases of adamantinoma were
identified in a review of the world literature published by Moon and
Mori in 1986.11
Osteofibrous dysplasia of long bone is also a rare lesion, but, in
contrast to adamantinoma, it is benign.12
Certain
clinicopathological features, however, such as the radiographic
appearance, a proclivity for the tibia, and an association with
fibro-osseous tissues are similar in both adamantinoma and osteofibrous
dysplasia, supporting the hypothesis of a relationship between
these two entities. An adamantinoma subtype has been described
("differentiated adamantinoma") which occurs intracortically in the
first two decades of life and is characterized histologically by a
predominantly osteofibrous dysplasia-like pattern and a small
inconspicuous component of epithelial elements scattered within the
stromal fibroblastic tissue.13
Classic adamantinoma
typically affects patients older than 20 years and exhibits variable
histological patterns to include basaloid, spindle, tubular, and
squamoid. Czerniak et al13
proposed the existence of a
continuum of fibro-osseous lesions with osteofibrous dysplasia at one
end of the spectrum, classic adamantinoma at the other, and
differentiated adamantinoma intermediately.
Cytogenetic analysis has contributed greatly to the understanding of
the histopathogenesis of many types of neoplasia. Adamantinoma and
osteofibrous dysplasia are of unknown histopathogenesis. Unfortunately,
because of their rarity, cytogenetic reports concerning these tumors
are few.2, 3, 4, 5, 6, 7
The karyotypic findings of only five cases of
classic adamantinoma and two cases of differentiated adamantinoma have
been described,2, 3, 4
Table 1
. An examination of the four current cases and a review of those
previously reported reveal that extra copies of chromosomes 7, 8, 12,
19, and/or 21 have been seen in all but one case of classic
adamantinoma and one case of differentiated adamantinoma. Extra copies
of one or more of these same chromosomes with the exception of
chromosome 19 have also been seen in osteofibrous dysplasia (Table 1)
,
lending further support to an osteofibrous dysplasia/adamantinoma
relationship.4, 5
It will be interesting to see if future studies of osteofibrous
dysplasia will also eventually reveal extra copies of chromosome 19, or
if this anomaly will remain confined to adamantinoma. Also, it should
be noted that structural anomalies to include translocations,
deletions, inversions, and marker chromosomes have been detected in
both differentiated and classic adamantinomas but not osteofibrous
dysplasia. The latter observation suggests that adamantinomas are
slightly more karyotypically complex than osteofibrous dysplasia. Often
these structural changes are in addition to the common numerical
changes (+7, +8, +12, and +21) seen in both adamantinoma and
osteofibrous dysplasia. It could be hypothesized that the expansion of
the abnormal clone to include structural changes parallels a
progression of an osteofibrous dysplasia lesion to an adamantinoma. The
observation that adamantinoma may develop from osteofibrous dysplasia
has also been supported by others.1, 5
It was unfortunate
that fresh tissue was not submitted for conventional karyotypic
analysis on the progressive lesions of the tibia and fibula of Case 4,
as it would have been interesting to compare the findings of each
lesion.
In summary, these cytogenetic and molecular cytogenetic data reveal
that extra copies of chromosomes 7, 8, 12, 19, and 21 are recurrent in
adamantinoma. Adamantinoma must be distinguished from a variety of
epithelial and soft tissue neoplasms. In particular, epithelial
elements of the basaloid, tubular, or squamoid patterns can be
misdiagnosed as metastatic carcinoma in a small biopsy specimen.
Dominant spindle-cell or small tubular patterns may be confused with a
fibrosarcoma or vascular neoplasm. Identification of these recurrent
genetic imbalances may ultimately prove to be a useful diagnostic
adjunct in adamantinoma.
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Acknowledgments
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We thank Kimberly Christian for expert secretarial service and
Marilu Nelson, Diane Pickering, Patty Cattano, and Michelle Hess for
expert technical assistance.
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Footnotes
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Address reprint requests to Julia A. Bridge, M.D., Department of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, NE 68198-3135. E-mail: jbridge{at}unmc.edu
Supported in part by grants from the Orthopaedic Research
Education Foundation and the John A. Wiebe, Jr. Children’s Health Care
Fund.
Accepted for publication November 22, 2000.
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References
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Dorfman HD, Czerniak B: Bone Tumors. St. Louis, Mosby, Inc., 1998, pp 949–973
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Mandahl N, Heim S, Rydholm A, Willen H, Mitelman F: Structural chromosome aberrations in an adamantinoma. Cancer Genet Cytogenet 1989, 42:187-190[Medline]
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Sozzi G, Miozzo M, Palma SD, Minelli A, Calderone C, Danesino C, Pastorino U, Pierotti MA, Porta GD: Involvement of the region 13q14 in a patient with adamantinoma of the long bones. Hum Genet 1990, 85:513-515[Medline]
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Hazelbag HM, Wessels JW, Mollevangers P, Van den Berg E, Molenaar WM, Hogendoorn PCW: Cytogenetic analysis of adamantinoma of long bones: further indications for a common histogenesis with osteofibrous dysplasia. Cancer Genet Cytogenet 1997, 97:5-11[Medline]
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Bridge JA, Dembinski A, DeBoer J, Travis J, Neff JR: Clonal chromosomal abnormalities in osteofibrous dysplasia: implications for histopathogenesis and its relationship with adamantinoma. Cancer 1994, 73:1746-1752[Medline]
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Bridge JA, Swarts SJ, Buresh C, Nelson M, Degenhardt JM, Spanier S, Maale G, Meloni A, Lynch JC, Neff JR: Trisomies 8 and 20 characterize a subgroup of benign fibrous lesions arising in both soft tissue and bone. Am J Pathol 1999, 154:729-733[Abstract/Free Full Text]
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Tarkkanen M, Kaipainen A, Karaharju E, Bohling T, Szymanska J, Helio H, Kivioja A, Elomaa I, Knuutila S: Cytogenetic study of 249 consecutive patients examined for a bone tumor. Cancer Genet Cytogenet 1993, 68:1-21[Medline]
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ISCN: An International System for Human Cytogenetic Nomenclature. Edited by Mitelman F. Basel, Switzerland, Karger, 1995
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Chan AS, Squire JA, Thorner P, Zielenska M: Molecular genetic changes in alveolar soft part sarcoma. Pediatr Pathol Mol Med 2000, 18:529-543
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Maier C: Ein primers myelogenes Platten-epithelkarzinom der Ulna. Beitraege zur klinischen Chirurgie 1900, 26:553-566
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Moon NF, Mori H: Adamantinoma of the appendicular skeleton: updated. Clin Orthop 1986, 204:215-237
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Campanacci M: Osteofibrous dysplasia of long bones: a new clinical entity. Ital J Orthop Traumatol 1976, 2:221-237[Medline]
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Czerniak B, Rojas-Corona RR, Dorfman HD: Morphologic diversity of long bone adamantinoma: the concept of differentiated (regressing) adamantinoma and relationship to osteofibrous dysplasia. Cancer 1989, 64:2319-2334[Medline]
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Abstract
Introduction
