Utility of P63 in Differentiating Giant Cell Tumor from Other Giant Cell-Containing Lesions
Monalisa HUI1, Shantveer G UPPIN1, K Karun KUMAR1, S RADHIKA2, P CHANDRASEKHAR3, K Nageshwara RAO3
1Department of Pathology, Nizam’s Institute of Medical Sciences, HYDERABAD, INDIA
2Department of Clinical Pharmacology and Therapeutics, Nizam’s Institute of Medical Sciences, HYDERABAD, INDIA
3Department of Orthopaedics, Nizam’s Institute of Medical Sciences, HYDERABAD, INDIA
Keywords: Giant cell tumor of bone, Giant cell-containing lesions of bone, p63, Immunohistochemistry
To assess P63 expression in giant cell-containing lesions of the bone (GCLB) and to determine its utility in differentiating giant cell
tumor of the bone (GCTB) from other GCLBs.
Material and Method: Cases diagnosed as GCLB on histopathology were included in the study. P63 immunohistochemistry was performed in
all the cases. The percentage of cells showing nuclear positivity was assessed in the non-giant cell component. Statistical analysis was performed
using the Mann-Whitney U test.
Results: Of the total 53 cases studied, the majority were GCTBs (23), followed by 12 cases of chondroblastomas (CBL) and 18 other giant cell
lesions (GCLs). All giant cell-containing lesions except one case of CBL and brown tumor of hyperparathyroidism (BTH) showed P63 staining
in the non-giant cell component. However, the mean P63 labeling of GCT (52.6%) was higher compared to CBL (28.3%), aneurysmal bone cyst
(ABC) (15.2%), non-ossifying fibroma (NOF) (24.5%), giant cell lesion of small bones (GCLSB) (11%), BTH (6.8%) and chondromyxoid fibroma
(CMF) (12.3%), with a p-value of <0.001.
Conclusion: Although p63 was present in majority of the GCLBs, its percentage positivity was significantly higher in GCTB compared to the
other GCLBs. The diagnosis of GCTB is likely if cut-off value of >50% is applied.
Morphology in correlation with clinical and radiological
findings is the cornerstone for the diagnosis of primary
bone tumors. The giant cell rich tumors of the bone are
morphologically distinct entities which share in common
the presence of multinucleated osteoclast-like giant cells.
. With the advent of minimally invasive procedures,
the material obtained for initial diagnosis of primary bone
tumors is often limited and poses a diagnostic dilemma.
Though routine morphology is sufficient in most of the
cases, immunohistochemistry (IHC) helps to resolve the
diagnostic difficulties that are especially encountered in
small biopsies with atypical morphology and ambiguous
imaging. Until the advent of anti-histone antibodies, there
was no well-established diagnostic marker for giant cell
tumor of the bone (GCTB). Studies have shown conflicting
results regarding over expression of p63 by IHC and
molecular methods in the stromal cells of GCTB 2-5
In this article we have assessed the expression of p63 in
giant cell-containing lesions of the bone and determined
its utility in differentiating GCTB from other giant cellcontaining
lesions of the bone (GCLBs).
The study included non-consecutive histologically verified
cases of various giant cell-containing lesions of the bone
(GCLB) where paraffin blocks were available for IHC.
The clinical features, location and imaging findings were
retrieved from the medical records. The diagnosis was
made on 42 curettage specimens, 6 open biopsies and 5
resected specimens. The hematoxylin and eosin-stained
sections of all the cases were reviewed along with the
clinical, imaging and other relevant laboratory findings
to confirm the original diagnosis. The appropriate
paraffin block was selected for IHC after examining the
representative hematoxylin and eosin-stained sections.
The decalcified sections, and areas of hemorrhage and
necrosis were excluded. IHC was performed on 3-4μm
thick sections using mouse monoclonal antibody against
p63 (Pre-diluted, Ready to Use Antibody, Biogenex).The
percentage of nuclear positivity was assessed in non-giant
cell component after counting a minimum of 500 nuclei
in the hot spots. The intensity of staining was evaluated as
weak (+1), moderate (+2) and strong (+3). Moderate to
strong intensity nuclear staining in >1% of the cells was
considered positive. Scoring was applied by two pathologists independently and the average of the two scores was taken
into account. IHC was performed in batches and slides with
a positive control were included in every batch. Statistical
analysis was performed using the Mann-Whitney U test,
and a p-value of <0.05 was considered significant. A
receiver operating characteristic (ROC) curve analysis was
done to determine the cut-off value of p-63 positivity in
order to predict the diagnosis of GCTB. Both the tests were
done using SPSS software version 20.
Of the total number of 53 cases studied, the majority were
GCTBs (23), followed by 12 cases of chondroblastoma
(CBL). The other GCLBs studied included 6 aneurysmal
bone cysts (ABC), 3 cases of non-ossifying fibroma
(NOF), in addition to 2 cases each from brown tumor of
hyperparathyroidism (BTH), giant cell lesion of small
bones (GCLSB) and chondromyxoid fibroma (CMF) and a 1 case each of giant cell rich reparative granuloma (GCRG),
osteoblastoma and telangiectatic osteosarcoma.
Regarding the 23 GCTBs, the age of the patients ranged
from 14 to 69 years with a mean age of 30.18 years. There
was a slight male predominance with a M:F ratio of 1.3:1.
The presentation was with pain and swelling in the distal
femur and proximal tibia in 18 patients, the distal radius
in 2 patients and one case each of the base of proximal
phalanx of the right ring finger, the left third metacarpal
and the proximal humerus. The plain radiographs of GCTB
involving various sites are illustrated in Figure 1(A-D). The
duration of the symptoms ranged from one month to 18
months. On histopathology, all showed a characteristic
biphasic pattern with spatial arrangement of the osteoclast
giant cells amidst the mononuclear cells as shown in Figure
1(E and F). The nuclei of the mononuclear cells resembled
the giant cells, which were large and had 40 to 50 nuclei.
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|Figure 1: Plain radiographs of giant cell tumor of bone (GCTB) presenting as expansile lytic lesions (white arrows) involving the epiphysis
of A) distal femur, B) proximal tibia, C) distal radius and D) first metatarsal. E, F) Histological sections of GCTB showing spatial
distribution of osteoclast-like giant cells and mononuclear cells (H&E; E; x200, F; x400). G, H) Diffuse strong nuclear p63 staining in the
mononuclear cells with sparing the nuclei of osteoclast-like giant cells (p63 antibody; G; x200, H; x400).
There was no clustering of giant cells. Osteoid formation
was not seen. Aneurysmal bone cyst-like changes were
noted in 7 cases. However, benign fibrous histiocytomalike
areas were not seen in any of the cases.
Regarding the 12 CBLs, the age of the patients ranged
from 12 to 35 years with a mean age of 18.1 years and M:F
of 1.4:1. The majority were located in the distal femur (4
cases) followed by the proximal tibia (3 cases) and the
proximal femur (2 cases). There was one case each located
in the distal fibula, calcaneum and manubrium sterni. The
duration of the symptoms ranged from 2.5 months to 3
years. On histopathology, the osteoclast-like giant cells were
randomly distributed. The mononuclear cells were uniform
round to polygonal with well-defined cytoplasmic borders
and longitudinal nuclear grooves. Pink hyaline cartilage and pericellular lace-like chicken wire calcifications were
also noted. Aneurysmal bone cyst-like changes was noted
in 2 cases. The plain radiographs and histopathological
findings of CBL are illustrated in Figure 2(A-F).
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|Figure 2: A, B) Plain radiographs of Chondroblastomas (CBL) showing a well-defined lytic lesions involving epiphysis of distal femur
and proximal tibia. C-F) Histological sections of CBL showing lobules of eosinophilic cartilaginous matrix with intervening cellular
areas. These cellular areas show sheets of polygonal shaped chondroblasts and osteoclast-like giant cells. On higher magnification
the chondroblasts have round oval vesicular which show indentations and longitudinal grooves. Areas of pericellular chicken-wire
calcification can be noted in (F). (H&E; C; x100, D; x400, E; x1000, F; x400). G, H) Moderate intensity nuclear staining for p63 in some
of the mononuclear cells (p63 antibody; x400)
The mean age of the ABC patients was 21 years and the
lesions were primarily located in the humerus (3 cases),
the vertebral bodies (2 cases) and the proximal femur (1
case). On microscopy, there were blood filled cystic spaces
separated by fibrous septae containing osteoclast-like giant
cells and proliferation of fibroblasts along with reactive
woven bone rimmed by osteoblasts. The three NOF
patients presented with a lytic lesion in the tibia and femur.
The two cases of giant cell lesion of the small bones (fourth
metacarpal and middle phalanx of the right middle finger)
are now considered as solid ABC whereas the term GCRG of jaw (1 Case) is still retained as it is in the recent World
Health Organization classification of soft tissue and bone
6. The giant cells showed clustering with fewer nuclei
as opposed to the uniform distribution of the giant cells
in GCTB. Both the cases of CMF were located in the left
tibia. The two cases of BTH were located in the mandible
and left tibia. These patients had elevated serum calcium and parathormone levels and were later found to have
parathyroid adenomas. The imaging and histopathological
findings of various giant cell-containing lesions are shown
in Figure 3(A, B, D, E, G, H, J and K). A single case of
osteoblastoma was located in the L4 vertebral body and a
case of telangiectatic variant of osteosarcoma involved the
left occipital bone.
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|Figure 3: A) Plain radiograph of chondromyxoid fibroma (CMF) showing well defined expansile eccentric lytic lesion involving
proximal metaphysis of tibia. B) Histological section of CMF showing lobules with central hypocellular and peripheral cellular areas
with chondromyxoid matrix in the background. Central areas contain bland spindle to stellate cells in a chondromyxoid background.
The peripheral areas show plump spindle to polygonal cells along with few osteoclast-like giant cells. (H&E; x100). C) Weak p63 staining
is seen in scattered cells. D) MRI spine of aneurysmal bone cyst reveals an expansile osteolytic lesion involving posterior elements of
vertebra with classic internal AIR-fluid levels. E) Histological section of ABC showing cystic space separated by septa containing bland
spindle cells and osteoclast-like giant cells (H&E; x100). F) Weak nuclear staining for p63 in scattered spindle cells. (p63 antibody; x
400). G) Plain radiograph of giant cell lesion of small bone (now considered to be solid ABC) presenting as well defined lytic lesion in
the metacarpal bone. H) Histological sections showing non-uniformly distributed giant cells amongst bland spindle shaped cells along
with focal haemorrhage (H&E; x100). I) Weak staining for p63 noted in few cells while rest of the cells are negative (p63 antibody; x400).
J) Plain radiograph of brown tumor of hyperparathyroidism (BTH) presenting as lytic lesion in the centre of mandible; K) Histological
section showing non-uniformly distributed giant cells with haemorrhage and fibrosis (H&E; x100). L) Most cells are negative for p63,
with only weak staining in scattered cells. (p63 antibody; x400).
All the GCTBs showed strong nuclear positivity in
the stromal cells and are depicted in Figure 1(G and
H). The percentage positivity of cells displaying p63
immunostaining ranged from 50.5% to 71 % except for one
case located in the distal femur that had a positivity of 14%.
None of the cases showed any evidence of nuclear staining
in the multinucleate giant cells. All the other GCLBs except
one case each of CBL and BTH showed p63 staining in
the non-giant cell component/ stromal cells. Out of the 11
cases of CBL that were positive for p63, 9 cases had weak
to moderate intensity staining in less than 50% of the cells
as shown in Figure 2(G and H). The mean p63 labeling in
GCTB (56.2%) was much higher compared to CBL (28.3%),
ABC (15.2%), NOF (24.5%), GCLSB (11%), BTH (6.8%)
and CMF (12.3%). A single case each of osteoblastoma,
GCRG and telangiectatic osteosarcoma showed nuclear
staining in 52.5%, 45% and 34.5% of the cells respectively.
The p63 positivity was found to be statistically significant in patients with GCTB when compared to non-GCTB as
analyzed by the Mann-Whitney U test (U=46.5, p<0.001).
ROC analysis showed a cut off value of 49.75 for p63 and
had a sensitivity of 95% and specificity of 90% to diagnose
GCTBs with an area under curve (AUC) of 93.3%, p <0.001.
The staining of p63 in CMF, ABC, GCLSB and BTH are
shown in Figure 3C, 3F, 3I and 3L respectively. The location
and distribution of p63 positive staining cells in GCTB and
various GCLBs are provided in Table I.
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|Table I: Distribution and p-63 staining of all the giant cell-containing lesions
GCLBs are a heterogeneous group of tumors and tumorlike
lesions of the bone with a wide range of differential
diagnosis. Definite diagnosis is challenging in the setting of
limited sampling, unusual age and location at presentation.
The morphology of the mononuclear cells gives a clue to
the diagnosis. However, the key diagnostic component
may be under represented in biopsy. Secondary changes like ABC which frequently accompanies GCTB or CBL
may obscure original morphology and overshadow the
underlying primary tumor in biopsy specimens 7,8
This study showed p63 expression in all cases (23/23) of
GCTB. Almost all the cases except one showed more than
50% nuclear positivity. The intensity of the staining was
strong and was limited to the mononuclear cells. Similar
to our study, Hammas, Dickson and Linden also reported
overexpression of p63 in all GCTB 2,9,10. De La Rosa G,
Paula and Lee reported p63 overexpression in 86.9%, 82%
and 81% of the cases respectively 3-5. Yanagisawa reported
higher mean p63-positivity for recurrent GCTB (73.6%)
compared to non-recurrent cases (29.1%) 11. However,
its usefulness as a prognostic marker in recurrence has not
been evaluated in our study.
Studies have shown variable expression of p63 in CBL
ranging from 30% to 83.3% 2-5. Dickson found expression
of p63 in 30% of the cases with a mild to moderate staining
intensity in 7-75 % of the cells 2. Although De la Roza
observed p63 expression in 10 out of 12 cases, the intensity
of staining was weak to moderate except in one case 4.
In contrast to strong nuclear staining observed in GCTB,
a weak to moderate intensity staining involving less than
50% cells were seen in 9 out of the 11 cases of CBL that
showed p63 positivity. The rate of p63 positivity in ABC
was much higher compared to the findings of Hammas
(40%), Lee (20%), Dickson (28.6%), Paula (51%) and De la
Roza (62.5%) 2-5,10.
Although GCTB affects a relatively older population, there
is often considerable overlap between the clinical features
of GCTB and CBL. GCT has also been documented in
children and adolescents with biological behavior similar to
that seen in adults, except a marked female predominance.
The presence of an open physis does not impede the tumor
to involve the epiphyseal cartilage 12. On the other hand,
CBL in adults more frequently involves the flat bones and
short bones of the hands/feet with an aggressive behavior
compared to children 13. As both the tumors are located
in the epiphyseal region, absence of a chondroid matrix
often causes confusion. To differentiate the above entities,
Lee recommends the use of S100 along with p63. A strong
nuclear p63 staining with weak S100 in the mononuclear
cells favors GCTB over CBL 3. Akpalo reported DOG1 as
a highly sensitive and specific marker for CBL 14.
The other giant cell-containing lesions like ABC, NOF,
GCLSB, and BTH showed positivity for p63 in all cases
but percentage of positivity and intensity of staining was
significantly lower than that of GCTB involving less than
50% of the cells. Expression of p63 in most of the GCLBs
may lower its specificity as a diagnostic marker. Hence a 50% cut-off value can be used to improve the specificity
that would reliably distinguish GCTB from other GCLBs
after taking into consideration the age and location of the
tumors. A similar suggestion was also made in the Paula
The morphology of GCRG closely resembles BTH. A
careful clinical history of hyperparathyroidism helps in
differentiating these two entities. All the other studies
except De la Roza have shown negative immunostaining for
p63 in all the cases of central giant cell granuloma (CGCG)
reflecting a pathogenesis different from GCTB 2-4,10,15.
The latter has shown p63 positivity in all the four cases of
CGCG (4). The single case of CGCG in our study was also
positive for p63 but the proportion of cells stained were less
than 50%. The number of cases of GCRG, osteoblastoma
and telangiectatic osteosarcoma included were very low
and this is a limitation of this study.
There is disagreement amongst the various authors
regarding the utility of p63 as a diagnostic marker in
GCTB. De la Roza showed no difference in p63 positivity
by immunostaining among the giant-cell-rich lesions such
as GCTB and CBL 4. Our results were consistent with
the reports of Hammas, Lee, Paulo et al and Dickson and
we suggest its use as a diagnostic marker provided with
a cut off value of 50% 2,3,5,10. However Dickson and
Lee considered 5% and 10% of cells respectively for cut
off 2,3. On the other hand, de La Roza considered any
nuclear staining of p63 as positive 4. The discrepancies
in staining may be attributed to the antibody clones and
antigen retrieval methods. Gene expression profiling have
also substantiated the above findings with over expression
of p63 in the majority of GCTBs and only a minor fraction
of other GCLBs 2,3.
Recent studies have identified H3 histone family member
3A (H3F3A) (G34W/V/R/L) mutations in the majority
of GCTBs and H3 histone family member 3B (H3F3B)
(K36M) mutations in nearly all CBLs, but these are absent
in other GCLBs. IHC using mutation-specific H3G34W
and H3K36M antibodies is highly specific for GCTB and
CBL respectively and can be used as a diagnostic tool in
limited biopsies 16-19. The presence of alternate H3F3A
mutations on Sanger sequencing further enhances the
diagnostic yield in a subset of GCTB which are negative
for H3G34W on IHC 20. The majority of primary ABCs
harbor clonal rearrangements of the USP6 gene locus.
Cases without the USP6 gene rearrangement hint at the
presence of morphologically undetected components of
GCTB and CBL in small biopsies 21. However, these novel
diagnostic techniques require expertise, standardization
and validation which are not feasible in the setting of
limited resources and are presently not widely available.
It is also important to differentiate GCTB from other GCLBs
as Denosumab has specific therapeutic implications for
GCTB and radiofrequency ablation for CBL. These can be
used as treatment options alternative to surgical resections
Though P63 expression can be seen to a variable extent
in all GCLBs of the bone, the percentage of positivity in
GCTB is significantly high compared to other GCLBs.
Hence p63 staining by IHC with a cut-off of 50% can be
used as an additional marker to differentiate GCTB from
other GCLBs of bone.
CONFLICT of INTEREST
The authors have no conflict of interest.
Manuscript preparation, literature search: MH, Concept,
definition of intellectual content, data analysis: SGU,
Data analysis: KK&SR, Manuscript review, supervision:
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