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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 3  |  Page : 617-629
Anti-Histone H3.3 G34W antibody is a sensitive and highly specific immunohistochemistry marker for the diagnosis of Giant cell tumor of bone. A validation based on analysis of 198 cases from a single centre in India


1 Department of Pathology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana, India
2 Department of Orthopaedics, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana, India
3 Department of Radiology and Imaging, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana, India
4 Department of Orthopedic Oncosurgery, Apollo Hospital and Udai Omni Hospital, Hyderabad, Telangana, India

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Date of Submission09-Mar-2021
Date of Decision22-Nov-2021
Date of Acceptance22-Dec-2021
Date of Web Publication21-Jul-2022
 

   Abstract 


Context: The diagnosis of giant cell tumor of bone (GCTB) is difficult in small biopsies with unusual age of presentation, location, and extensive secondary changes. Most of the GCTBs harbor H3F3A G34W mutations with a subset of cases showing alternate G34V, G34R, and G34L mutations. Objectives: To analyze the expression of anti-histone H3.3G34W antibody in different cellular components of GCTB across different locations and presentations (including the unusual ones) and validate the utility of this antibody in the diagnosis of GCTB and differentiate it from the other osteoclast-like giant-cell-rich lesions. Design: Immunohistochemistry was performed using anti-histone H3.3G34W antibody in the diagnosed cases of GCTB (136 cases of GCTB from 133 patients, including two malignant GCTBs) and other giant cell-containing lesions (62 cases). The presence of unequivocal crisp nuclear staining was considered positive. Results: Immunohistochemistry revealed unequivocal nuclear positivity in the mononuclear cells in 87.3% of the cases of GCTB. Of these, most showed diffuse expression with moderate to strong intensity staining. The positive staining was restricted to the nuclei of mononuclear cells with the nuclei of osteoclastic giant cells being distinctly negative. In addition to conventional GCTBs, two cases each of multicentric and malignant GCTB showed positive staining. The other giant-cell containing lesions were distinctly negative. The present study showed a sensitivity of 87.3% with specificity and positive predictive value of 100%. Conclusion: The anti-histone G34W antibody is a highly sensitive and specific marker for the diagnosis of GCTB and differentiating it from its mimics. The positive staining is restricted to the mononuclear cell component of GCTB with sparing the osteoclastic giant cells further reiterating the fact that the mononuclear stromal cells are the true neoplastic component of GCTB.

Keywords: Anti-histone H3.3G34W antibody, giant cell tumor of bone, immunohistochemistry

How to cite this article:
Kamble A, Hui M, Rao K N, Narayanan R, Reddy B R, Uppin SG, Chandrasekhar P. Anti-Histone H3.3 G34W antibody is a sensitive and highly specific immunohistochemistry marker for the diagnosis of Giant cell tumor of bone. A validation based on analysis of 198 cases from a single centre in India. Indian J Pathol Microbiol 2022;65:617-29

How to cite this URL:
Kamble A, Hui M, Rao K N, Narayanan R, Reddy B R, Uppin SG, Chandrasekhar P. Anti-Histone H3.3 G34W antibody is a sensitive and highly specific immunohistochemistry marker for the diagnosis of Giant cell tumor of bone. A validation based on analysis of 198 cases from a single centre in India. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 15];65:617-29. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/617/351597





   Introduction Top


The giant cell tumor of bone (GCTB) accounts for 4–5% of the primary bone tumors. It is composed of the proliferation of mononuclear stromal cells and large osteoclastic giant cells. The mononuclear cells are of two types, macrophage-like osteoclast precursors and stromal cells that express receptor activator of the nuclear factor kappa β ligand.[1] Though the osteoclast precursor and the true neoplastic stromal cells are morphologically indistinguishable, immunohistochemically, the former are CD14- and CD61-positive and the latter are negative. The stromal cells which are negative for CD14 and CD61 harbor histone mutations and represent the neoplastic component of GCTB.[2] The GCTB does not pose any major diagnostic challenge in classical clinical and radiologic settings. However, difficulty arises in situations like unusual age of presentation, unusual location, multicentricity, increased mitotic activity, extensive areas of the aneurysmal bone cyst (ABC)-like change, exuberant benign fibrous histiocytoma (BFH)-like areas, infarction, post-denosumab therapy, and others. The challenges become amplified in small biopsies making the definitive diagnosis difficult.

Recently, somatic driver mutations in the H3 histone family members 3A (H3F3A) and 3B (H3F3B) have been identified in 92% GCTB and 95% chondroblastoma (CBL), respectively.[2] Around 85–95% of the GCTBs harbor H3F3A G34W mutations. In a subset of cases, alternate G34V, G34R, and G34L mutations have been noted.[3],[4],[5],[6] The introduction of monoclonal mutation-specific antibodies directed against the mutant H3.3G34W and H3.3K36M proteins have revolutionized ancillary diagnostic modalities in bone tumors.[7],[8]

Currently, no studies are demonstrating similar mutations in GCTBs affecting the Indian population. The present study was designed to demonstrate the utility of the anti-histone H3F3AG34W antibody in the diagnosis of GCTB and its usefulness in differentiating it from other osteoclastic giant-cell-rich lesions. In this article, we have analyzed its expression in different cellular components of GCTB across different locations and presentations (including the unusual ones). The expression of this marker in various other giant-cell-containing lesions mimicking GCTB was also analyzed.


   Materials and Methods Top


This study was performed on the archival tissue available in the form of formalin-fixed paraffin-embedded (FFPE) tissue. This study was approved by the Institutional Ethics Committee. The diagnosed cases of GCTB and other giant cell-containing lesions where the FFPE tissue was available for immunohistochemistry (IHC) were included in the study. The majority of the cases included were GCTB (136 cases of GCTB from 133 patients, including two malignant GCTBs). The remaining 62 cases were other giant cell-containing lesions which are listed in [Table 1] from an equal number of patients.
Table 1: Distribution of various giant cell-containing lesions of the bone

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The relevant demographic, clinical features, imaging, and other laboratory findings were retrieved from the hospital and departmental medical records. The site of the tumor, multicentricity (if any), and the location of the tumor within the bone (epiphysis/metaphysis/diaphysis) were noted. The specimens submitted for the histological study included biopsies (64), curettage specimens (86), resection (35), and amputation (13).

All the specimens had been processed for routine paraffin sections after fixation in 10% neutral buffered formalin. Decalcification was carried out with 10% nitric acid whenever necessary before processing. After confirmation of the morphology on hematoxylin and eosin (H and E)-stained sections, the representative blocks were selected for IHC study, excluding the decalcified sections, necrotic, and hemorrhagic areas wherever possible.

Immunohistochemistry

Immunohistochemistry was performed on 3–4 micron thick sections using anti-histone rabbit monoclonal antibody H3F3A (H3.3) G34W (clone RM 263, Medasys Livermore, CA, USA; dilution 1:100) on Roche Ventana Benchmark GX fully-automated platform. A case of conventional GCTB with classic location and imaging showing positive staining on IHC was the chosen as control. The presence of unequivocal crisp nuclear staining with H3.3G34W in the cells irrespective of the intensity and proportion was considered as positive staining.


   Results Top


A total of 198 cases were analyzed for the expression of H3.3 G34W. These included 136 cases of GCTB from 133 patients and 62 cases of other giant cell-containing lesions.

Giant cell tumor of the bone (n = 133 patients)

The age of the patients ranged from 10 to 65 years with a mean age of 31.1 years and a median of 29 years. There was a slight male preponderance with a M: F ratio of 1.2:1. Most of the patients had presented with pain associated with/without swelling. A pathological fracture was noted in six patients. A history of trivial trauma was noted in three patients; of the 133 patients, 116 had presented with primary tumor and the remaining 17 were recurrent. There were two unusual cases of multicentric GCTB and two cases of malignant GCTB (secondary).

A total of 144 sites were affected in 133 patients and the distribution of these is shown in [Figure 1]a. Most of them involved the appendicular skeleton (123, 85.4%), predominantly ends of long bones. Multicentricity was noted in two patients. In one patient, five metachronous lesions and one recurrence were documented over 11 years. The affected sites included the fifth metacarpal bone of the right hand, the intermediate cuneiform bone of the right foot, the left proximal humerus, lateral malleolus of the right tibia, and right distal femur. The patient had a recurrence of the right distal tibial lesion within 2 years. Another patient presented with recurrent GCTB of the left distal femur and proximal tibia who was operated on with curettage and bone grafting twice in the past [Figure 3]a. On the positron emission tomography (PET) scan, additional lytic lesions were detected in the left iliac bone, head of the second metatarsal of the left foot, the body of sternum, multiple vertebrae, proximal femur, and left talus. Both the patients had normal serum calcium, phosphorus, and parathormone levels ruling out the possibility of brown tumor of hyperparathyroidism. The two cases of malignant GCTB involved proximal tibia and proximal humerus, respectively. One of them was a 30-year-old female who presented with recurrence and pulmonary metastasis 1 year following a limb salvage surgery. Another patient was a 39-year-old male who developed malignant transformation at his second recurrence 27 months after the initial diagnosis. Following the initial diagnosis of GCTB, he had undergone curettage and 1 year later developed the first recurrence. This was again treated with re-curettage and allogeneic bone grafting. Both these patients did not receive any radiotherapy.
Figure 1: (a) Site distribution of giant cell tumors analyzed. (b) Site distribution of chondroblastoma and aneurysmal bone cyst (including giant cell lesion of small bones) [ () – CBL; [ ] – ABC; { } – GCLSB]. (c- l) Radiographs of GCTBs affecting different sites that tested positive for anti-histone H3.3G34W. All of these mainly presented as geographic lytic lesions with narrow zone transition affecting (c) the distal right femur, (d) proximal right tibia, (e) distal right radius, (f) proximal right femur, (g) proximal left humerus (h) body and glenoid process of left scapula, (i) proximal phalanx of the third digit of the right hand, (j) roof of the acetabulum of the right iliac bone, (k) sacrum and (l) proximal metaphysis of the right tibia. In the tubular bones, these mainly affected the epi- metaphyseal region extending to the subchondral region except one of the purely metaphyseal/metadiaphyseal lesion (l). There is no evidence of matrix calcifications, periosteal reaction, or soft-tissue component any of the lesions depicted. A cortical break can be seen in lesions involving (c) the distal femur, (g) proximal humerus, and (j) acetabulum

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Figure 2: (a) and (b) Histological section of GCTB showing mononuclear cells and osteoclastic giant cells in characteristic spatial distribution. Note the similarities between the nuclei of the giant cells and adjacent mononuclear cells [H and E; A ×200, B ×400]. (c) Blood-filled cystic spaces separated by the septa-containing spindle cells and giant cells representing ABC-like change in GCTB [H and E; ×400]. (d) Area of BFH-like change in a GCTB showing a storiform arrangement of the spindle cells admixed with scattered giant cells [H and E; ×100]. (e-j) Immunohistochemistry with anti-histone H3.3 G34W antibody showing nuclear positivity within the mononuclear cells of GCTB with sparing of the nuclei of adjacent osteoclastic giant cells. (e) and (f) Diffuse strong staining in the mononuclear cells [E ×200, F ×400]. (g) Diffuse moderate intensity of staining [×400]. (h) Focal weak staining [×1000]. (i) Positive staining in the nuclei of some of the mononuclear cells within the septa of ABC-like areas within GCTB [×400]. (j) Positive staining in the nuclei of some spindle cells in BFH-like area of the GCTB [×400]

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Figure 3: (a–c) Case of multicentric GCTB. (a) Plain radiographs showing geographic osteolytic lesions with a narrow zone of transition involving the distal femur and proximal tibia. There are no matrix calcifications, periosteal reaction, or soft-tissue components. (b) A histological section showing classical morphology of GCTB with spatially arranged mononuclear cells and osteoclastic giant cells [H and E; ×400]. (c) Diffuse nuclear positivity for anti-histone H3.3 G34W antibody in the mononuclear cells [X400]. (d–f) Case of GCTB initially diagnosed as ABC. (d) Plain radiograph shows eccentric expansile osteolytic lesions involving the distal femur reaching up to the subarticular area. The matrix shows faint internal septations. No periosteal reaction or soft-tissue component. (e) Histological sections show septa-containing spindle cells, giant cells, and osteoid (buckled) separating the cystic spaces [H and E; ×200]. (f) Nuclear positivity for anti-histone H3.3 G34W antibody in the mononuclear cells lining and within the septa [×400]. (g–l) Case of secondary malignant GCTB. (g and h) Sections showing spindle to polygonal cells with pleomorphic hyperchromatic nuclei and moderate to abundant eosinophilic cytoplasm. Area of necrosis and increased mitoses including the atypical ones can also be noted [H and E; G, H ×200]. (i) Area of osteoid formation within the lesion [H and E; ×200]. (j) Focal residual conventional GCTB areas showing bland mononuclear cells admixed with osteoclastic giant cells [H and E; X200]. (k) and (l) Positive nuclear staining for anti-histone H3.3 G34W both within the sarcomatous and residual conventional GCTB components [k and l ×400]

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All the lesions were osteolytic involving either the epiphyseal/epi-metaphyseal region, except two cases, that involved the proximal metadiaphyseal region of the tibia [Figure 1]. Additional fluid-fluid levels were reported in eight cases in which the differentials included primary ABC versus GCTB with ABC-like change.

On histopathology, the tumors showed a characteristic biphasic pattern with the spatial arrangement of the osteoclastic giant cells amid the mononuclear cells. There was no clustering of giant cells [Figure 2]a and [Figure 2]b. Some cases showed reparative bone formation at the periphery Microscopically, ABC-like change was noted in 32 cases and BFH-like areas in 15 cases [Figure 2]c and [Figure 2]d. One case with extensive ABC-like areas without identifiable GCTB component, initially reported as primary ABC was later revised as GCTB with extensive ABC-like change after IHC results and review of clinical, imaging, and microscopic findings [Figure 3]d, [Figure 3]e, [Figure 3]f. All the lesions of multicentric and metadiaphyseal GCTB had classic morphology of the conventional type [Figure 3]b.

Both the cases of secondary malignant GCTB predominantly showed sheets of spindle to polygonal mononuclear cells with atypia and frequent mitoses including the atypical ones. In addition, there were large areas of necrosis with focal osteoid matrix formation. Of these two patients, one underwent above-knee amputation, which also showed similar findings with lesions extending up to the subcutaneous tissue [Figure 3]g, [Figure 3]h, [Figure 3]i. Focal residual conventional GCTB areas with bland morphology were also noted in both cases [Figure 3]j. In both cases, the initial core biopsies and curettage specimens (done twice in the second case) had only conventional GCTB without any sarcomatous component. Hence, a diagnosis of secondary malignant GCTB with osteosarcomatous sarcoma component was made.

Immunohistochemistry

IHC was performed in 136 samples taken from 133 GCTB patients. Both the primary and recurrent tumors were tested in two patients and one patient with multicentric GCTB—lesion from two different sites was tested. For analysis purposes, both the primary and recurrent tumors tested from the same individual were counted as one.

Immunohistochemistry using anti-histone antibody H3.3G34W revealed unequivocal nuclear positivity in the mononuclear cells in 117/134 (87.3%) cases. Of these, the majority (112/117, 95.7%) showed diffuse expression with moderate to strong intensity in the mononuclear cells [Figure 2]e, [Figure 3]f, [Figure 3]g. Focal weak positivity was noted in five cases [Figure 2]h. The positive staining was restricted to the nuclei of mononuclear cells with the nuclei of osteoclastic giant cells being distinctly negative. Positive-stained cells were also noted within ABC-like and BFH-like areas though relatively fewer in number [Figure 2]i and [Figure 2]j.

One case with an initial diagnosis of primary ABC had shown distinct nuclear positivity of anti-histone H3.3G34W antibody within some of the mononuclear cells in the septa [Figure 3]f. On subsequent review of the imaging findings, the lesion was seen extending to the subarticular region of the distal femur. Hence, a revised diagnosis of GCTB with extensive ABC-like change was made.

All the three tumors from patients with multicentric GCTBs (two tumors from one patient and one from the second patient) showed positive staining [Figure 3]c. Of the two cases located in the unusual metadiaphyseal region, one case showed crisp nuclear positivity and the other was negative. The two cases of malignant GCTB showed positive nuclear staining in both the sarcomatous as well as residual conventional GCTB areas [Figure 3]k and [Figure 3]l.

Seventeen cases of GCTB were negative for anti-histone antibody H3.3G34W on IHC. All cases except two involved classic locations of the epiphysis/epi-metaphysis of the long bones. Of the remaining two, one was located in the proximal metadiaphyseal region of the tibia and the other involved the phalangeal bone. All these cases had a classical morphology of conventional GCTB with additional ABC-like change in 10 and BFH-like areas in 1. The cases of GCTB which were negative on IHC are summarized in [Table 2].
Table 2: Cases of GCTB negative on IHC

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Other giant cell-containing lesions (n = 62)

The CBLs (n = 26) were most commonly located in the distal femur followed by the proximal tibia, proximal humerus, and talus. The other sites affected included the maxilla, acetabulum, and temporal bone [Figure 1]b. All the cases of CBL included had classical morphology and ABC-like changes were seen in five cases [Figure 4]a, [Figure 4]b, [Figure 4]c. There were five cases of primary ABC, two of them involving the tibia, and one each affecting the femur, humerus, and ulna. All the cases showed blood-filled cystic spaces separated by the septa of variable thickness. The septa were lined by spindle cells and osteoclastic giant cells. Reactive woven bone was noted along the contour of the septa [Figure 4]e, [Figure 4]f, [Figure 4]g, [Figure 4]h. There were five cases of giant cell lesions of the small bone (GCLSB) which are now called the solid variants of ABC. Histopathology in these cases had shown mononuclear cells and irregularly distributed osteoclastic giant cells [Figure 4]j and [Figure 4]k. The demographic features, location, and histopathological findings of other giant cell-containing lesions included in the study are summarized in [Table 3] and [Figure 4]m and n and [Figure 5] and [Figure 6].
Table 3: Demographic features, location, and histopathological findings of other giant cell-containing lesions

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Figure 4: (a–d) Chondroblastoma. (a) Radiograph shows a geographic lytic lesion with a narrow zone of transition involving the distal epiphysis of the femur. No matrix calcification, periosteal reaction, or soft-tissue component. (b and c) The histological sections show areas of eosinophilic cartilaginous matrix and adjacent cellular areas with the admixture of mononuclear cells and osteoclastic giant cells. The mononuclear cells are polygonal in shape with round to oval nuclei and a moderate amount of eosinophilic cytoplasm. The nuclei of these cells show indentations and longitudinal grooves [H and E; b × 100, c ×1000]. (d) Negative staining for anti-histone H3.3 G34W within the tumor cells [X400]. (e–i) Aneurysmal bone cyst. (e) Radiograph of the pelvis including both the hip joints shows a geographic lytic lesion with multiple internal septations and narrow zone of transition involving the upper end of the left femur. There is a thin uninterrupted periosteal reaction along its medial cortex with e/o soft-tissue component. (f) Axial T2-weighted image of the pelvis at the level of lesser tuberosity showing an expansile lytic lesion with multiple “fluid-fluid” levels in the left femur. (g and h) Histological sections show blood-filled cystic spaces separated by septa. These septa contain bland spindle cells, osteoclastic giant cells along with newly-laid osteoid. [H and E; g × 100, h ×200]. (i) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (j–l) Giant cell lesion of the small bones of the hands and feet now considered solid ABC. (j) Plain radiographs showing an expansile osteolytic lesion with a narrow zone of transition involving the middle phalanx of the third digit of the left hand. The matrix of the lesion shows no calcification. No periosteal reaction or soft-tissue component is seen. (k) Histological section showing fibroblastic bland spindle cells admixed with irregularly distributed multinucleated giant cells with adjacent areas of hemorrhage [H and E; X200]. (l) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (m-o) Brown tumor of hyperparathyroidism. (m) Radiograph shows a large soft-tissue density swelling in the mandibular region, causing smooth scalloping along the outer cortex of the mandible. It shows no calcification. (n) Histological section shows variable admixture of spindle fibroblastic cells, irregularly distributed (clustering around areas of hemorrhage) multinucleated osteoclastic giant and osteoid [H and E; X100]. (o) Negative staining for anti-histone H3.3 G34W within the lesional cells [×400]

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Figure 5: (a–d) Chondromyxoid fibroma. (a) Radiograph shows an eccentric multilobulated geographic lytic lesion with a narrow zone of transition and sclerotic margins involving the lower meta-diaphysis left tibia. Cortical buttressing is seen along the superior aspect of the lesion. No matrix calcification, periosteal reaction, or soft-tissue component is seen. (b and c) Histological sections show lobules of the chondromyxoid matrix with central hypocellular and peripheral cellular areas. The central hypocellular areas contain bland spindle to stellate cells in abundant chondromyxoid matrix. The peripheral cellular areas contain spindle cells, chondroblasts, and multinucleated giant cells [H and E; b × 100, c × 200]. (d) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (e-g) Osteoid osteoma. (e) Radiograph shows an eccentric mildly expansile lytic lesion with a narrow zone of transition and sclerotic margins involving the proximal phalanx of the second digit of the right hand. The matrix of the lesion shows central calcification. No periosteal reaction or soft-tissue component is seen. (f) Histological section showing irregular interconnecting bony trabeculae rimmed by osteoblasts with intervening vascularized fibrous stroma along with scattered giant cells [H and E; X100]. (g) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (h–j) Osteoblastoma. (h) Axial CT shows a lytic lesion with a narrow zone of transition, dense sclerotic margins, and cortical hypertrophy involving the right lamina of the lumbar vertebra. The matrix of the lesion shows central calcification. No soft-tissue component is seen. (i) Histological section showing interconnecting irregular trabeculae of osteoid with intervening osteoblasts admixed with scattered giant cells [H and E; X200]. (j) Negative staining for anti-histone H3.3G34W within the lesional cells [X400]. (k–m) Solitary bone cyst. (k) Radiograph shows a geographic lytic lesion with a narrow zone of transition, marginal sclerosis in the proximal meta-diaphysis (intertrochanteric) region of the left femur. A cortical break is seen along the superior aspect of the lesion. No matrix calcifications or soft-tissue components. (l) Histological section showing cyst wall containing fibrous tissue, newly-laid osteoid, and scattered giant cells [H and E; X200]. (m) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]

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Figure 6: (a and b) Gnathic giant cell reparative granuloma. (a) Histological sections show variable admixture of spindled fibroblastic cells, irregularly distributed multinucleated osteoclast-like giant cells [H and E; X200]. (b) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (c and d) Non-ossifying fibroma. (d) Histological sections show a storiform arrangement of bland spindle cells with a few admixed multinucleated osteoclastic giant cells [H and E; X200]. (d) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (e and f) Tenosynovial giant cell tumor. (e) Histological sections show a lesion with a variable admixture of oval to spindle mononuclear cells, multinucleated osteoclastic giant, and hyalinized collagen [H and E; X200]. (f) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (g–j) Giant cell-rich osteosarcoma. (g and h) Radiograph shows a geographic lytic lesion with a wide zone of transition involving the distal right tibia. A cortical break is seen along the medial aspect of the lesion with soft-tissue component. No periosteal reaction or matrix calcifications. (i) Histological sections show admixture osteoclastic giant cells and atypical mononuclear cells. Apart from pleomorphism and hyperchromasia, increased mitotic activity including atypical ones can be seen within the mononuclear cells. [X400]. (j) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (k–n) Telangiectatic osteosarcoma. (k) Radiograph shows a mixed lytic-sclerotic lesion with a wide zone of transition involving the proximal tibia. A cortical break is seen along the medial aspect of the lesion with sunburst periosteal reaction and soft-tissue component. (l) T2WI of MRI of the knee joint sagittal view shows a mixed intensity lesion in the proximal tibia with central hyperintense areas and peripheral iso-intense areas. (m) Histological sections show cystic spaces separated by the septa. The septa show the admixture of osteoclastic giant cells and atypical mononuclear cells [H and E; X100]. (n) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400]. (o–q) Clear-cell chondrosarcoma. (o) Radiograph shows a geographic lytic lesion with a wide zone of transition, extending up to the subchondral region involving the proximal humerus. The lesion does not show any matrix calcification, cortical break, or soft-tissue component. (p) Histological section shows sheets of polygonal tumor cells with round to oval nuclei and abundant clear cytoplasm with a distinct cell membrane. Prominent metaplastic bony trabeculae can be noted within the lesion [H and E; X200]. (q) Negative staining for anti-histone H3.3 G34W within the lesional cells [X400].

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Immunohistochemistry

All the other giant cell-containing lesions except GCTB showed negative immunostaining in both the mononuclear cells and osteoclastic giant cells [Figure 4]d, [Figure 4]i, [Figure 4]l, [Figure 4]o, and [Figure 5] and [Figure 6].

Of the total 136 cases of GCTB, 117 were positive for anti-histone H3.3G34W IHC denoting a sensitivity of 87.3%. None of the other giant cell-containing lesions were positive on IHC indicating a specificity of 100%. The positive predictive value was 100% and the negative predictive value was 78.49% (95% CI: 68.71–84.13%).


   Discussion Top


The giant cell-rich lesions of the bone incorporate a diverse group of tumor and tumor-like lesions of the bone. GCTB is frequently located in the epi-metaphyseal region of the long bones. Metadiaphyseal location without the involvement of the epiphysis is rare.[9],[10],[11],[12],[13] In this study, two cases involved the metadiaphyseal region of the proximal tibia.

The small bones of the hands and feet are rare sites of involvement in GCTB ranging from 1.2 to 4% in the hand and 0.9 to 1.8% in the foot.[14] In the present study, eight cases affecting the small bones of hands and feet were included. Giant cell tumors of bone are usually solitary and less than 1% of the cases are multicentric.[15] Multicentric lesions tend to develop more commonly in unusual locations like small bones of the hand and feet.[16],[17] In the two cases of multicentric GCTBs included in the present study, the lesions affected both the long bones and small bones of the hands and feet. One of these cases has been published earlier.[18]

In the literature, ABC-like change and BFH-like areas are reported in 14 and 12% of all the GCTBs.[19],[20] Among the cases of GCTB included in the study, 23.9% had ABC-like changes. Though the presence of ABC-like changes does not severely impact the treatment and outcome of GCTBs, however, selective sampling of the cystic areas in biopsy leads to diagnostic dilemma.[21] The BFH-like areas resemble non-ossifying fibroma (NOF) with subtle differences. According to the recent World Health Organization classification of bone tumors, the BFH located in the epiphysis represents GCTB (with regressive changes) while those situated in the metaphysis should be referred to as NOF.[1]

Malignant GCTB (earlier term malignancy in GCTB) is rare accounting for less than 10% of all GCTBs and can be either primary or secondary.[22],[23] The sarcomatous component can show varied morphology including osteosarcoma, as was noted in both the cases included in the present study. The closest differential diagnosis includes giant cell-rich osteosarcoma.

Until recently there were no specific IHC or molecular markers for either GCTBs or other giant cell-rich lesions. Hence, their differentiation was solely based on the morphology in correlation with the clinic-radiology findings. Some of the studies in the past have shown p63 overexpression in the mononuclear cells of GCTB implying its utility as a prospective diagnostic marker.[24],[25],[26],[27],[28] Though Dickson et al. and Hammas et al. reported p63 overexpression in all GCTBs, De La Roza and Lee found immunoreactivity in 86.9 and 81% of the GCTBs, respectively. The above studies have also shown variable staining on IHC in ABC and CBL.[24],[25],[26],[28] Due to the lack of specificity, the use of this IHC marker in differentiating various giant cell-containing lesions of the bone is limited.

In recent times, a mutation has been identified in GCTB affecting the H3F3A gene coding for the histone variant H3.3 (H3.3-G34W).[29] Of the total 235 GCTB analyzed by Amary et al.,[8] 90.6% of the cases were positive on IHC with anti-histone H3.3G34W. On further sequencing, the sensitivity of H3.3G34 mutation increased to 94.8% after excluding the cases which were non-informative on genetics. In the present study, the sensitivity of this IHC marker for GCTB was found to be 87.3%, which was similar to Schaefer et al.[3] but slightly lower than Amary et al.[8] The specificity of this marker in all the studies including the present one was 100%. So IHC with anti-histone H3.3G34W antibody can be used as a novel diagnostic marker for the diagnosis of GCTB, especially in laboratories where high-end molecular techniques are not available.

Both the cases of multicentric GCTB tested were positive and in one of the patients, two distinct lesions tested were also positive. This suggests that multicentric GCTBs harbor histone mutation identical to conventional solitary GCTBs. Hence, IHC with anti-histone H3.3G34W will be a valuable adjunct not only for the diagnosis of these rare multicentric GCTBs but also to differentiate these from other multicentric giant cell-rich lesions, especially brown tumors of hyperparathyroidism.

A subset of GCTB negative on IHC may still harbor H3.3G34W detectable by sequencing. The remaining may either have alternate H3F3A mutations (G34V, G34L, and G34M) or maybe wild-type.[8] In the present study, there were 17 such negative cases on IHC. These were not subjected to sequencing. So, the diagnosis of GCTBs should not be completely ruled out even if IHC is negative especially in cases with classical clinical, radiological, and pathological findings. However, if feasible, such cases should ideally be subjected to genotyping of the H3.3 mutation. This is especially true for cases with overlapping features with CBL and ABC. IHC with mutant-specific antibody H3.3 K36M/genotyping for H3.3K36M and fluorescent in situ hybridization (FISH) analysis for USP6 gene rearrangement should be done to rule out the above entities, respectively.

The alternate H3.3 variants (G34L, M, and V) most commonly involve the small bones of the hands and feet, patella, and axial skeleton.[8] In the present study, most of the negative cases affected the long bones and all the axial lesions included were positive. Among the negative cases, the one involving the phalanx and the other affecting the metadiaphyseal region of the tibia need genotyping for H3.3 to confirm the diagnosis. If found negative, USP6 rearrangement should be tested to rule out the possibility of ABC.

Behjati et al.[2] reported H3.3 p.G34 mutations in 2% of osteosarcoma with G34R and G34W substitution. The deoxyribonucleic acid (DNA) methylation profile of these H3.3F mutant tumors is more closely related to GCTB and differs from the wild-type osteosarcomas. So, the authors extrapolated that these tumors signify the malignant counterpart of GCTB instead of true osteosarcomas.[3],[8],[30] The detection of H3F3A mutations by H3G34W IHC and/or by sequencing in malignant tumors arising from GCTB helps in differentiating malignant GCTB and osteosarcoma. In the present study, both the cases of malignant GCTB showed positivity in both sarcomatous and focally residual conventional GCTB areas.

Studies have shown contradictory results with some showing retained nuclear expression and others demonstrating loss of protein in the malignant counterpart.[5],[31],[32] Amary et al.[8] noted positivity in 2.85% of the cases of osteosarcoma. Of these, one was malignant GCTB with a low-grade osteosarcomatous component, and the rest were osteosarcoma with osteoclast-rich component and chondroblastic osteosarcoma. All three cases of osteosarcoma (giant cell-rich-1 and telangiectatic-2) and one case of clear-cell chondrosarcoma tested in the present study were negative.

The most common differential diagnosis of GCTB is CBL. The diagnostic difficulty is compounded if the calcified chondroid matrix of CBL is absent on imaging or not sampled in small biopsies. To differentiate the above entities, Lee et al.[25] advocated the use of a combination of p63 and S100. A strong nuclear p63 staining with weak S100 in the mononuclear cells favors GCTB over CBL. Akpalo et al.[33] reported Discovered on gastrointestinal stromal tumor-1 (DOG-1) IHC as a sensitive and specific marker for IHC. On the contrary, Cleven et al.[34] reported low sensitivity of 33%. H3K36M IHC has been found to be highly sensitive and specific for the diagnosis of CBL.[3],[8] Similar to Amary et al.,[8] IHC with anti-histone H3.3 G34W antibody was negative in all the cases of CBL included in the present study.

All the cases of ABCs including the five giant cell-lesions of the small bones were negative on IHC except for one case revised later as GCTB which has already been discussed above along with GCTB. The finding of positively stained cells within the septa should alert one to the possibility of GCTB with extensive ABC-like areas masquerading as primary ABC. It will be diagnostically very useful while dealing with small biopsies. It also obviates the need to perform FISH studies for USP6 rearrangement to confirm ABC, especially in resource-poor settings.

All the remaining giant cell-containing lesions were negative on IHC with anti-histone H3.3G34W antibody. These results are consistent with other studies.[8] The limitations of the present study include (i) lack of testing for all mutant proteins, (ii) negative cases were not subjected to sequencing for G34W and variant mutations, and (iii) some of the GCTB mimics included were few.

To conclude, the findings in the present study provide strong evidence that the anti-histone G34W antibody is a highly sensitive and absolutely specific marker for the diagnosis of GCTB and differentiating it from its mimics. The positive staining is restricted to the mononuclear cell component of GCTB with sparing the osteoclastic giant cells, further reiterating the fact that the mononuclear stromal cells are the true neoplastic component of GCTB. This study not only demonstrates positive staining in the GCTBs located across different sites but also in a few cases of multicentric GCTB and two cases of malignant GCTBs tested. None of the other giant cell lesions included in the study tested positive, making the marker specific to GCTB.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Shantveer G Uppin
Department of Pathology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_265_21

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