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PTHrP receptor in cartilaginous tumours: a marker for malignancy?
Pathology (2002 ) 34, pp. 133– 137
ANATOMICAL
PATHOLOGY
Co-expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in cartilaginous tumours: a marker for malignancy? TOSHIYUKI KUNISADA*, JANE M. MOSELEY‡, JOHN L. SLAVIN†, T. JOHN MARTIN‡ PETER F. M. CHOONG*
AND
Departments of *Orthopaedics and †Pathology, St Vincent’s Hospital, and ‡St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
Summary Aim: Parathyroid hormone-related protein (PTHrP) is one of the critical factors for the differentiation and growth of chondrocytes. We examined the correlation between the co-expression of PTHrP and PTH/PTHrP receptor and the grade of malignancy in cartilaginous tumours. Methods: We analysed PTHrP and PTH/PTHrP receptor expression in chondrosarcoma by immunohistochemistr y and compared specific staining with the expression in benign cartilaginous tumours. There were 38 cartilaginous bone tumours consisting of 26 chondrosarcoma, six enchondroma and six osteochondroma. Chondrosarcom a were composed of 20 conventional chondrosarcom a (10 grade 1, seven grade 2, and three grade 3), two dedifferentiated chondrosarcoma, two clear cell chondrosarcoma, and two myxoid chondrosarcoma. We performed the standard peroxidase-labelled streptavidin –biotin detection method for immunohistochemistry using an antibody raised against PTHrP (1–14) and PTH/PTHrP receptor. The magnitude of receptor positivity of PTHrP and PTH/ PTHrP in each tumour was assessed as a percentage of PTHrP and PTH/PTHrP-positive cells per thousand tumour cells in the most histologically aggressive area of the tumour. Results: All chondrosarcoma, five of six enchondroma, and four of six osteochondroma showed PTHrP-positive cells, and all chondrosarcoma, five of six enchondroma and five of six osteochondroma showed PTHrP receptor-positive cells. The grade of malignancy correlated with the percentage of both PTHrP and PTH/PTHrP receptor-positive tumour cells (P < 0.0001, either). Each grade of chondrosarcoma showed statistically higher expression of both PTHrP and PTH/PTHrP receptor than benign cartilaginous tumour. Conclusion: This is the first report of the co-expression of PTHrP and PTH/PTHrP receptor in chondrosarcoma . PTHrP and PTH/PTHrP receptor positivity may be valuable for differentiating between benign and malignant cartilaginous tumours. Key words: Parathyroid hormone-related protein ( PTHrP), PTH/PTHrP receptor, chondrosarcoma, progression. Received 14 December 2000, revised 18 September, accepted 24 September 2001
INTRODUCTION Chondrosarcoma is a malignant tumour of cartilage, in which the matrix formed by tumour cells is uniformly and entirely chondroid in nature.1 Histological grading for this tumour indicates a wide range of malignancy,2 and chondrosarcoma is classed into three malignancy grades depending on the cellularity of tumour and the cytological atypia.3 However, the intratumoural histological heterogeneity makes grading of chondrosarcoma difficult. This is particularly true when trying to differentiate between grade 1 and grade 2 lesions, and occasionally between benign and grade 1 lesions. This is important because of the differences in treatment strategies, not only between benign and grade 1 tumours but also between grade 1 and high grade ( grade 2 and 3) lesions. Apart from the combination of histological features and imaging, no other diagnostic modalities are routinely cited for grading chondrosarcoma, although several papers have reported features that are related to grade and progression of chondrosarcoma, such as type of collagen expression,4 mutation of oncogenes,5,6 and collagenase expression.7,8 Parathyroid hormone-related protein ( PTHrP) is associated with humoral hypercalcaemia of malignancy9 and its expression has been demonstrated in a large number of tumour types and tumour-derived cell lines.10 Recently, many papers have implicated PTHrP production in tumour cells with progression of tumour malignancy,11,12 although it remains unclear what effect PTHrP production has on tumour cells. However, there is little doubt that PTHrP production promotes the growth of metastatic tumour cells in bone.13 PTHrP is also expressed in wide range of normal cells including cartilage,10 and it has an important developmental role in endochondral bone formation.14 Recent studies of mice null for the PTHrP gene demonstrated that PTHrP not only slows the differentiation of chondrocyte in the growth plate but also controls endochondral bone formation,15 through its activities via PTH/PTHrP receptor ( PTH type 1 receptor).16 However, there are few reports of PTHrP production in cartilaginous tumours. Despite the importance of PTHrP to our understanding of the biology of chondrosarcoma, PTH/PTHrP receptor has not been assessed in chondrosarcoma nor has its expression been related to clinical and histopathological factors. In the present study, we have examined the co-expression of PTHrP and PTH/PTHrP receptor in cartilaginous tumours
ISSN 0031–3025 printed/ISSN 1465– 3931 online/02/020133 – 05 © 2002 Royal College of Pathologists of Australasia DOI:10.1080/003130201201117936
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Fig. 1 Immunohistochemistry of PTHrP and its receptor are shown: a case with grade 1 chondrosarcoma showing 50% PTHrP-positive cells ( A) and 75% PTHrP receptor ( B) in three fields averaged across tumour; Grade 2 chondrosarcoma, 70% PTHrP positivity ( C) and 95% PTHrP receptor positivity ( D); Grade 3 chondrosarcoma, 100% PTHrP positivity ( E) and 100% PTHrP receptor positivity ( F). No signal was observed with NIRS as negative control of PTHrP ( G) nor without primary antibody as negative control of PTH/PTHrP receptor ( H) in grade 3 chondrosarcoma ( bars = 50 mm).
PTHrP AND PTH/PTHrP RECEPTOR EXPRESSION IN CHONDROSARCOMA
and discussed its value in determining the grade of malignancy.
MATERIALS AND METHODS Archival samples of 38 cartilaginous tumours consisting of 26 chondrosarcoma, six enchondroma and six osteochondroma were used. These specimens were from 18 males and 20 females with a median age of 44 years ( range 15–76 years). All specimens were diagnosed and examined to score the grade of chondrosarcoma by a single pathologist ( JLS). Chondrosarcoma was composed of 20 conventional chondrosarcoma ( 10 grade 1, seven grade 2 and three grade 3), two dedifferentiated chondrosarcoma, two grade 1 clear cell chondrosarcoma and two myxoid chondrosarcoma ( one grade 2 and one grade 3). Immunohistochemistry PTHrP We performed the standard peroxidase-labelled streptavidin–biotin detection method as previously described for PTHrP.17 Briefly, all specimens were fixed in cold 10% buffered formalin, processed and embedded in paraffin. Five-mm-thick sections were cut and immunostained with a rabbit polyclonal antibody against N-terminal human PTHrP ( 1–14). Incubation of sections with 1 in 500 of the primary antiserum was carried out overnight at 4°C in a humidified chamber. The peroxidase activity was detected with 3,39-diaminobenzidine tetrahydrochloride ( Sigma, USA) and 0.15% hydrogen peroxidase. Slides were counterstained with haematoxylin, dehydrated and mounted on a coverslip. Each examination included a positive tissue control (skin) and substitution of the primary antiserum with non-immune rabbit serum ( NIRS) at the same dilution as a negative control.
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expressed as a percentage of the total. After this assessment, 20 of these slides were randomly selected and reviewed by JLS. This review showed concurrence of assessment in 10 slides, difference within 5% in eight, and difference within 10% in two. Following this, the results were compared with the grade of malignancy. Statistical analysis Correlations between continuous variables and between continuous and ordinal variables were estimated using Spearman rank correlation. Statistical differences between each group of ordinal variables were calculated using the Scheffe’s F test. All statistical analyses were carried out using computer software Statview 4.5 ( Abacus Concepts, USA). Test results with P < 0.05 were regarded as significant.
PTH/PTHrP receptor PTH/PTHrP receptor was identified also by similar streptavidin–peroxidase enzyme conjugate method using the mouse monoclonal antibody MMS-610P to PTH/PTHrP receptor ( Covance, USA). The primary antibody was applied at a dilution of 1 in 150 and incubated at 4°C overnight. Each examination included a positive tissue control and a negative control prepared by omission of the primary antibody.
RESULTS Both PTHrP and PTH/PTHrP receptor immunoreactivity were detected predominantly in the cytoplasm of the tumour cells ( Fig. 1). Some nuclei of the tumour cells occasionally showed positive staining for both PTHrP and PTH/PTHrP receptor. There was a clear gradation in the intensity of stained cells, which stained either strongly or weakly, and this variation was occasionally present throughout the same tumour. PTHrP and PTH/PTHrP receptor positivity are summarised in Table 1. All chondrosarcoma, five of six enchondroma and four of six osteochondroma demonstrated PTHrP-positive cells. All chondrosarcoma, five of six enchondroma and five of six osteochondroma showed PTH/ PTHrP receptor-positive cells. Average PTHrP and PTH/ PTHrP receptor-positive rates are shown in Table 2. Both PTHrP and PTH/PTHrP receptor-positive rates significantly correlated with the grade of malignancy of cartilaginous tumours ( P < 0.0001, either), indicating higher positivity for both PTHrP and PTH/PTHrP receptor in higher grades of the tumour. Each grade of chondrosarcoma showed statistically higher expression of both PTHrP and PTH/PTHrP receptor than benign tumour. PTHrP positivity also correlated with PTH/PTHrP receptor positivity in the same tumour (adjusted R 2 = 0.71, P < 0.0001 ).
Evaluation of immunohistochemical expression A histological section was prepared under supervision by our sarcoma pathologist ( JLS) from the most aggressive part of each tumour. Each slide was chosen after screening several H&E-stained sections taken from the tissue block, with the aim of utilising a slide that was most representative of the tumour, according to Choong et al. 18 One of us ( TK) examined the entire specimen of each slide without knowledge of the histological diagnosis (benign or malignant) and the grade of malignancy. PTHrP and PTH/PTHrP receptor-positive cells were counted and determined as the percentage of 1000 tumour cells in three different locations of one section which represented more positive cells and stronger staining. Intra- and inter-tumour heterogeneity was addressed by regarding each stained cell as positive regardless of staining intensity and negative when there was absence of staining in the non-immune or negative controls. The average number of stained cells at the three sites was
DISCUSSION Recent papers reported that PTHrP could be one of most critical molecules for chondrocyte differentiation. Analysis of PTHrP homozygous knockout mice showed that absence of PTHrP during embryonic development causes chondrodysplasia, 19,20 with histological examination revealing a diminution of chondrocyte proliferation and premature maturation of chondrocytes. Conversely, transgenic mice overexpressing PTHrP demonstrated delayed bone development with an accumulation of prehypertrophic chondrocytes.21,22 In vitro studies have also demonstrated that PTHrP inhibits matrix mineralisation and apoptosis of hypertrophic chondrocytes.23 In the growth plate, PTH/PTHrP receptor mediates the action of PTHrP to slow differentiation of chondrocytes, and the combined expression of PTHrP and PTH/PTHrP receptor
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KUNISADA et al.
TABLE 1 Summary of PTHrP and PTH/PTHrP receptor staining in cartilaginous tumours
No.
Grade
PTHrP ( %) *
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Benign Benign Benign Benign Benign Benign Benign Benign Benign Benign Benign Benign 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 Dedifferentiated Dedifferentiated
0 0 0 1 5 10 30 30 30 40 40 95 40 40 50 50 65 75 75 80 80 80 80 95 60 65 65 70 85 90 100 100 85 95 100 100 100 100
PTHrP receptor ( %) * 0 0 5 5 1 15 35 35 45 50 65 85 30 70 75 90 30 20 100 40 70 70 80 70 75 60 70 95 95 80 90 100 80 100 100 100 95 100
* PTHrP or PTH/PTHrP receptor positivity is expressed as percentage of positive staining cells in a area of 1000 tumour cells in each of three random locations on one representative section.
regulates chondrocyte and cartilage maturation.24,25 Thus, PTHrP and PTH/PTHrP receptor appear to maintain chondrocytes in a proliferative state, to slow the rate of chondrocyte maturation and to delay programmed cell death of the hypertrophic chondrocyte. Chondrosarcoma is a malignant tumour of cartilage, and cell lines derived from human chondrosarcoma have characteristics similar to chondrocytes.26,27 Our results showed that higher staining of PTHrP was noted in high grade chondrosarcoma and lower staining in benign tumours, which was consistent with the previous report of Amling et al. 28 Moreover, we have shown that the level of positivity of PTH/PTHrP receptor correlated with that of PTHrP. High grade chondrosarcoma grows aggressively, while benign cartilaginous tumour that is well-differentiated grows very slowly, sometimes remaining unchanged. Our results of high staining of PTHrP and PTH/PTHrP receptor in high grade tumour support the notion that PTHrP maintains chondrosarcoma cells in a high proliferative state and low differentiation, and that the converse in benign tumour cells is also true. Moreover, the close similarity between PTHrP and PTH/PTHrP receptor staining in high
TABLE 2 PTHrP and PTH/PTHrP receptor in benign and in malignant tumours, grade 1– 3 and dedifferentiated
Grade
n
Benign Grade 1 Grade 2 Grade 3 Dedifferentiated
12 12 8 4 2
PTHrP ( %) * 23.4 67.5 79.4 95.0 100.0
± ± ± ± ±
27.8 18.1 16.3 7.1 0.0
PTHrP receptor ( %) * 28.1 62.1 83.1 95.0 97.5
± ± ± ± ±
28.6 25.7 14.1 10.0 3.5
* PTHrP or PTH/ PTHrP receptor positivity is expressed as percentage of positive staining cells in a area of 1000 tumour cells in each of three random locations on one representative section.
grade tumour indicated that a similar factor could activate both expression of PTHrP and PTH/PTHrP receptor, since there was a strong correlation between the two in the same tumour. The differentiation between low grade chondrosarcom a and enchondroma can be difficult.3 This has an important impact on treatment modalities where benign tumours may be managed conservatively, while malignant lesions require surgical removal. In this series, the expression of both PTHrP and PTH/PTHrP receptor in chondrosarcoma was significantly higher than that in benign cartilaginous tumours, indicating that immunohistological staining of PTHrP and its receptor may be useful for differentiating between low grade chondrosarcoma and a benign enchondroma. For example, the mean positivity for grade 1 chondrosarcoma was three times higher than that of an enchondroma. In addition, a cut-off index of 45% PTHrP positivity in this study indicated a sensitivity of 92% (24 of 26) and a specificity of 92% ( 11 of 12), and a cut-off index of 70% PTH/PTHrP receptor positivity suggested a sensitivity of 88% (23 of 26) and a specificity of 92% (11 of 12). PTHrP and PTH/PTHrP receptor co-expression has been studied in several cancers,29–31 and it was reported that PTHrP could activate the growth of metastatic tumour cells in bone through interaction with the PTH/PTHrP receptor.32 Our results also suggest the possibility that PTHrP could activate the growth of chondrosarcoma cells in an autocrine/ paracrine way in bone tissue, in the same way that PTHrP activates the growth of metastatic cancer cells in bone. This is the first report of the co-expression of PTHrP and PTH/PTHrP receptor in chondrosarcoma. High expression of PTH/PTHrP receptor as well as PTHrP itself was detected in high grade chondrosarcoma, implicating these factors in the growth and progression of malignancy of chondrosarcoma. ACKNOWLEDGEMENTS We would like to thank Pat Smith, Elizabeth Williams and Toni Harris for their help with immunohistochemistry. Address for correspondence: Professor P. F. M. Choong, Department of Orthopaedics, St Vincent’s Hospital, Level 3 Daly Wing, 41 Victoria Parade, Fitzroy, Vic 3065, Australia. E-mail: [email protected]
References 1. Dorfman HD, Czerniak B. Bone tumours. St. Louis: Mosby, 1998; 353– 440. 2. Evans HL, Ayala AG, Romsdahl MM. Prognostic factors in chondrosarcoma of bone: a clinicopathologic analysis with emphasis on histologic grading. Cancer 1977; 40: 818– 31.
PTHrP AND PTH/PTHrP RECEPTOR EXPRESSION IN CHONDROSARCOMA
3. Unni KK. Dahlin’s bone tumour: general aspects and data on 11,087 cases. Philadelphia: Lippincott-Raven, 1996; 71–108. 4. Ueda Y, Oda Y, Tsuchiya H, Tomita K, Nakanishi I. Immunohistological study on collagenous proteins of benign and malignant human cartilaginous tumours of bone. Virchows Arch A Pathol Anat Histopathol 1990; 417: 291–7. 5. Wadayama B, Toguchida J, Yamaguchi T, Sasaki MS, Yamamuro T. p53 expression and its relationship to DNA alterations in bone and soft tissue sarcomas. Br J Cancer 1993; 68: 1134– 9. 6. Dobashi Y, Sugimura H, Sato A, et al. Possible association of p53 overexpression and mutation with high-grade chondrosarcoma. Diagn Mol Pathol 1993; 2: 257– 63. 7. Uria JA, Balbin M, Lopez JM, et al. Collagenase-3 ( MMP-13) expression in chondrosarcoma cells and its regulation by basic fibroblast growth factor. Am J Pathol 1998; 153: 91–101. 8. Scully SP, Berend KR, Toth A, Qi WN, Qi Z, Block JA. Interstitial collagenase gene expression correlates with in vitro invasion in human chondrosarcoma. Clin Orthop 2000; 376: 291– 303. 9. Suva LJ, Winslow GA, Wettenhall RE, et al. A parathyroid hormonerelated protein implicated in malignant hypercalcemia: cloning and expression. Science 1987; 237: 893–6. 10. Moseley JM, Gillespie MT. Parathyroid hormone-related protein. Crit Rev Clin Lab Sci 1995; 32: 299– 343. 11. Asadi F, Farraj M, Sharifi R, Malakouti S, Antar S, Kukreja S. Enhanced expression of parathyroid hormone-related protein in prostate cancer as compared with benign prostatic hyperplasia. Hum Pathol 1996; 27: 1319–23. 12. Luparello C, Birch MA, Gallagher JA, Burtis WJ. Clonal heterogene ity of the growth and invasive response of a human breast carcinoma cell line to parathyroid hormone-related peptide fragments. Carcinogenesis 1997; 18: 23–9. 13. Guise TA. Parathyroid hormone-related protein and bone metastases. Cancer 1997; 80: 1572– 80. 14. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin C J. Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 1996; 273: 613– 22. 15. Volk SW, Leboy PS. Regulating the regulators of chondrocyt e hypertroph y. J Bone Miner Res 1999; 14: 483– 6. 16. Mannstadt M, Juppner H, Gardella TJ. Receptors for PTH and PTHrP: their biological importance and functional properties. Am J Physiol 1999; 277: 665–75. 17. Kartsogiannis V, Moseley J, McKelvie B, et al. Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model. Bone 1997; 21: 385–92. 18. Choong PF, Akerman M, Willen H, et al. Prognostic value of Ki-67 expression in 182 soft tissue sarcomas. Proliferation – a marker of metastasis? APMIS 1994; 102: 915–24. 19. Amizuka N, Warshawsky H, Henderson JE, Goltzman D, Karaplis A
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C. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation. J Cell Biol 1994; 126: 1611– 23. Karaplis AC, Luz A, Glowacki J, et al. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev 1994; 8: 277–89. Amling M, Neff L, Tanaka S, et al. Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J Cell Biol 1997; 136: 205–13. Weir EC, Philbrick WM, Amling M, Neff LA, Baron R, Broadus AE. Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc Natl Acad Sci USA 1996; 93: 10240 –5. Zerega B, Cermelli S, Bianco P, Cancedda R, Cancedda FD. Parathyroid hormone [PTH( 1–34)] and parathyroid hormone-related protein [PTHrP( 1–34) ] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells. J Bone Miner Res 1999; 14: 1281– 9. Chung UI, Lanske B, Lee K, Li E, Kronenberg H. The parathyroid hormone /parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyt e differentiation. Proc Natl Acad Sci USA 1998; 95: 13030 – 5. Lanske B, Karaplis AC, Lee K, et al. PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 1996; 273: 663– 6. Kunisada T, Miyazaki M, Mihara K, et al. A new human chondrosarcoma cell line ( OUMS-27) that maintains chondrocytic differentiation. Int J Cancer 1998; 77: 854–9. Takigawa M, Tajima K, Pan HO, et al. Establishment of a clonal human chondrosarcoma cell line with cartilage phenotypes. Cancer Res 1989; 49: 3996– 4002. Amling M, Posl M, Hentz MW, Priemel M, Delling G. PTHrP and Bcl-2: essential regulatory molecules in chondrocyte differentiation and chondrogenic tumours. Verh Dtsch Ges Pathol 1998; 82: 160– 9. Iezzoni JC, Bruns ME, Frierson HF, et al. Coexpression of parathyroi d hormone-related protein and its receptor in breast carcinoma: a potential autocrine effector system. Mod Pathol 1998; 11: 265–70. Carron JA, Fraser WD, Gallagher JA. PTHrP and the PTH/PTHrP receptor are co-expressed in human breast and colon tumours. Br J Cancer 1997; 76: 1095– 8. Downey SE, Hoyland J, Freemont AJ, Knox F, Walls J, Bundred NJ. Expression of the receptor for parathyroid hormone-related protein in normal and malignant breast tissue. J Pathol 1997; 183: 212–7. Iddon J, Bundred NJ, Hoyland J, et al. Expression of parathyroid hormone-related protein and its receptor in bone metastases from prostate cancer. J Pathol 2000; 191: 170–4.
ANATOMICAL
PATHOLOGY
Co-expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in cartilaginous tumours: a marker for malignancy? TOSHIYUKI KUNISADA*, JANE M. MOSELEY‡, JOHN L. SLAVIN†, T. JOHN MARTIN‡ PETER F. M. CHOONG*
AND
Departments of *Orthopaedics and †Pathology, St Vincent’s Hospital, and ‡St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
Summary Aim: Parathyroid hormone-related protein (PTHrP) is one of the critical factors for the differentiation and growth of chondrocytes. We examined the correlation between the co-expression of PTHrP and PTH/PTHrP receptor and the grade of malignancy in cartilaginous tumours. Methods: We analysed PTHrP and PTH/PTHrP receptor expression in chondrosarcoma by immunohistochemistr y and compared specific staining with the expression in benign cartilaginous tumours. There were 38 cartilaginous bone tumours consisting of 26 chondrosarcoma, six enchondroma and six osteochondroma. Chondrosarcom a were composed of 20 conventional chondrosarcom a (10 grade 1, seven grade 2, and three grade 3), two dedifferentiated chondrosarcoma, two clear cell chondrosarcoma, and two myxoid chondrosarcoma. We performed the standard peroxidase-labelled streptavidin –biotin detection method for immunohistochemistry using an antibody raised against PTHrP (1–14) and PTH/PTHrP receptor. The magnitude of receptor positivity of PTHrP and PTH/ PTHrP in each tumour was assessed as a percentage of PTHrP and PTH/PTHrP-positive cells per thousand tumour cells in the most histologically aggressive area of the tumour. Results: All chondrosarcoma, five of six enchondroma, and four of six osteochondroma showed PTHrP-positive cells, and all chondrosarcoma, five of six enchondroma and five of six osteochondroma showed PTHrP receptor-positive cells. The grade of malignancy correlated with the percentage of both PTHrP and PTH/PTHrP receptor-positive tumour cells (P < 0.0001, either). Each grade of chondrosarcoma showed statistically higher expression of both PTHrP and PTH/PTHrP receptor than benign cartilaginous tumour. Conclusion: This is the first report of the co-expression of PTHrP and PTH/PTHrP receptor in chondrosarcoma . PTHrP and PTH/PTHrP receptor positivity may be valuable for differentiating between benign and malignant cartilaginous tumours. Key words: Parathyroid hormone-related protein ( PTHrP), PTH/PTHrP receptor, chondrosarcoma, progression. Received 14 December 2000, revised 18 September, accepted 24 September 2001
INTRODUCTION Chondrosarcoma is a malignant tumour of cartilage, in which the matrix formed by tumour cells is uniformly and entirely chondroid in nature.1 Histological grading for this tumour indicates a wide range of malignancy,2 and chondrosarcoma is classed into three malignancy grades depending on the cellularity of tumour and the cytological atypia.3 However, the intratumoural histological heterogeneity makes grading of chondrosarcoma difficult. This is particularly true when trying to differentiate between grade 1 and grade 2 lesions, and occasionally between benign and grade 1 lesions. This is important because of the differences in treatment strategies, not only between benign and grade 1 tumours but also between grade 1 and high grade ( grade 2 and 3) lesions. Apart from the combination of histological features and imaging, no other diagnostic modalities are routinely cited for grading chondrosarcoma, although several papers have reported features that are related to grade and progression of chondrosarcoma, such as type of collagen expression,4 mutation of oncogenes,5,6 and collagenase expression.7,8 Parathyroid hormone-related protein ( PTHrP) is associated with humoral hypercalcaemia of malignancy9 and its expression has been demonstrated in a large number of tumour types and tumour-derived cell lines.10 Recently, many papers have implicated PTHrP production in tumour cells with progression of tumour malignancy,11,12 although it remains unclear what effect PTHrP production has on tumour cells. However, there is little doubt that PTHrP production promotes the growth of metastatic tumour cells in bone.13 PTHrP is also expressed in wide range of normal cells including cartilage,10 and it has an important developmental role in endochondral bone formation.14 Recent studies of mice null for the PTHrP gene demonstrated that PTHrP not only slows the differentiation of chondrocyte in the growth plate but also controls endochondral bone formation,15 through its activities via PTH/PTHrP receptor ( PTH type 1 receptor).16 However, there are few reports of PTHrP production in cartilaginous tumours. Despite the importance of PTHrP to our understanding of the biology of chondrosarcoma, PTH/PTHrP receptor has not been assessed in chondrosarcoma nor has its expression been related to clinical and histopathological factors. In the present study, we have examined the co-expression of PTHrP and PTH/PTHrP receptor in cartilaginous tumours
ISSN 0031–3025 printed/ISSN 1465– 3931 online/02/020133 – 05 © 2002 Royal College of Pathologists of Australasia DOI:10.1080/003130201201117936
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Fig. 1 Immunohistochemistry of PTHrP and its receptor are shown: a case with grade 1 chondrosarcoma showing 50% PTHrP-positive cells ( A) and 75% PTHrP receptor ( B) in three fields averaged across tumour; Grade 2 chondrosarcoma, 70% PTHrP positivity ( C) and 95% PTHrP receptor positivity ( D); Grade 3 chondrosarcoma, 100% PTHrP positivity ( E) and 100% PTHrP receptor positivity ( F). No signal was observed with NIRS as negative control of PTHrP ( G) nor without primary antibody as negative control of PTH/PTHrP receptor ( H) in grade 3 chondrosarcoma ( bars = 50 mm).
PTHrP AND PTH/PTHrP RECEPTOR EXPRESSION IN CHONDROSARCOMA
and discussed its value in determining the grade of malignancy.
MATERIALS AND METHODS Archival samples of 38 cartilaginous tumours consisting of 26 chondrosarcoma, six enchondroma and six osteochondroma were used. These specimens were from 18 males and 20 females with a median age of 44 years ( range 15–76 years). All specimens were diagnosed and examined to score the grade of chondrosarcoma by a single pathologist ( JLS). Chondrosarcoma was composed of 20 conventional chondrosarcoma ( 10 grade 1, seven grade 2 and three grade 3), two dedifferentiated chondrosarcoma, two grade 1 clear cell chondrosarcoma and two myxoid chondrosarcoma ( one grade 2 and one grade 3). Immunohistochemistry PTHrP We performed the standard peroxidase-labelled streptavidin–biotin detection method as previously described for PTHrP.17 Briefly, all specimens were fixed in cold 10% buffered formalin, processed and embedded in paraffin. Five-mm-thick sections were cut and immunostained with a rabbit polyclonal antibody against N-terminal human PTHrP ( 1–14). Incubation of sections with 1 in 500 of the primary antiserum was carried out overnight at 4°C in a humidified chamber. The peroxidase activity was detected with 3,39-diaminobenzidine tetrahydrochloride ( Sigma, USA) and 0.15% hydrogen peroxidase. Slides were counterstained with haematoxylin, dehydrated and mounted on a coverslip. Each examination included a positive tissue control (skin) and substitution of the primary antiserum with non-immune rabbit serum ( NIRS) at the same dilution as a negative control.
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expressed as a percentage of the total. After this assessment, 20 of these slides were randomly selected and reviewed by JLS. This review showed concurrence of assessment in 10 slides, difference within 5% in eight, and difference within 10% in two. Following this, the results were compared with the grade of malignancy. Statistical analysis Correlations between continuous variables and between continuous and ordinal variables were estimated using Spearman rank correlation. Statistical differences between each group of ordinal variables were calculated using the Scheffe’s F test. All statistical analyses were carried out using computer software Statview 4.5 ( Abacus Concepts, USA). Test results with P < 0.05 were regarded as significant.
PTH/PTHrP receptor PTH/PTHrP receptor was identified also by similar streptavidin–peroxidase enzyme conjugate method using the mouse monoclonal antibody MMS-610P to PTH/PTHrP receptor ( Covance, USA). The primary antibody was applied at a dilution of 1 in 150 and incubated at 4°C overnight. Each examination included a positive tissue control and a negative control prepared by omission of the primary antibody.
RESULTS Both PTHrP and PTH/PTHrP receptor immunoreactivity were detected predominantly in the cytoplasm of the tumour cells ( Fig. 1). Some nuclei of the tumour cells occasionally showed positive staining for both PTHrP and PTH/PTHrP receptor. There was a clear gradation in the intensity of stained cells, which stained either strongly or weakly, and this variation was occasionally present throughout the same tumour. PTHrP and PTH/PTHrP receptor positivity are summarised in Table 1. All chondrosarcoma, five of six enchondroma and four of six osteochondroma demonstrated PTHrP-positive cells. All chondrosarcoma, five of six enchondroma and five of six osteochondroma showed PTH/ PTHrP receptor-positive cells. Average PTHrP and PTH/ PTHrP receptor-positive rates are shown in Table 2. Both PTHrP and PTH/PTHrP receptor-positive rates significantly correlated with the grade of malignancy of cartilaginous tumours ( P < 0.0001, either), indicating higher positivity for both PTHrP and PTH/PTHrP receptor in higher grades of the tumour. Each grade of chondrosarcoma showed statistically higher expression of both PTHrP and PTH/PTHrP receptor than benign tumour. PTHrP positivity also correlated with PTH/PTHrP receptor positivity in the same tumour (adjusted R 2 = 0.71, P < 0.0001 ).
Evaluation of immunohistochemical expression A histological section was prepared under supervision by our sarcoma pathologist ( JLS) from the most aggressive part of each tumour. Each slide was chosen after screening several H&E-stained sections taken from the tissue block, with the aim of utilising a slide that was most representative of the tumour, according to Choong et al. 18 One of us ( TK) examined the entire specimen of each slide without knowledge of the histological diagnosis (benign or malignant) and the grade of malignancy. PTHrP and PTH/PTHrP receptor-positive cells were counted and determined as the percentage of 1000 tumour cells in three different locations of one section which represented more positive cells and stronger staining. Intra- and inter-tumour heterogeneity was addressed by regarding each stained cell as positive regardless of staining intensity and negative when there was absence of staining in the non-immune or negative controls. The average number of stained cells at the three sites was
DISCUSSION Recent papers reported that PTHrP could be one of most critical molecules for chondrocyte differentiation. Analysis of PTHrP homozygous knockout mice showed that absence of PTHrP during embryonic development causes chondrodysplasia, 19,20 with histological examination revealing a diminution of chondrocyte proliferation and premature maturation of chondrocytes. Conversely, transgenic mice overexpressing PTHrP demonstrated delayed bone development with an accumulation of prehypertrophic chondrocytes.21,22 In vitro studies have also demonstrated that PTHrP inhibits matrix mineralisation and apoptosis of hypertrophic chondrocytes.23 In the growth plate, PTH/PTHrP receptor mediates the action of PTHrP to slow differentiation of chondrocytes, and the combined expression of PTHrP and PTH/PTHrP receptor
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TABLE 1 Summary of PTHrP and PTH/PTHrP receptor staining in cartilaginous tumours
No.
Grade
PTHrP ( %) *
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Benign Benign Benign Benign Benign Benign Benign Benign Benign Benign Benign Benign 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 Dedifferentiated Dedifferentiated
0 0 0 1 5 10 30 30 30 40 40 95 40 40 50 50 65 75 75 80 80 80 80 95 60 65 65 70 85 90 100 100 85 95 100 100 100 100
PTHrP receptor ( %) * 0 0 5 5 1 15 35 35 45 50 65 85 30 70 75 90 30 20 100 40 70 70 80 70 75 60 70 95 95 80 90 100 80 100 100 100 95 100
* PTHrP or PTH/PTHrP receptor positivity is expressed as percentage of positive staining cells in a area of 1000 tumour cells in each of three random locations on one representative section.
regulates chondrocyte and cartilage maturation.24,25 Thus, PTHrP and PTH/PTHrP receptor appear to maintain chondrocytes in a proliferative state, to slow the rate of chondrocyte maturation and to delay programmed cell death of the hypertrophic chondrocyte. Chondrosarcoma is a malignant tumour of cartilage, and cell lines derived from human chondrosarcoma have characteristics similar to chondrocytes.26,27 Our results showed that higher staining of PTHrP was noted in high grade chondrosarcoma and lower staining in benign tumours, which was consistent with the previous report of Amling et al. 28 Moreover, we have shown that the level of positivity of PTH/PTHrP receptor correlated with that of PTHrP. High grade chondrosarcoma grows aggressively, while benign cartilaginous tumour that is well-differentiated grows very slowly, sometimes remaining unchanged. Our results of high staining of PTHrP and PTH/PTHrP receptor in high grade tumour support the notion that PTHrP maintains chondrosarcoma cells in a high proliferative state and low differentiation, and that the converse in benign tumour cells is also true. Moreover, the close similarity between PTHrP and PTH/PTHrP receptor staining in high
TABLE 2 PTHrP and PTH/PTHrP receptor in benign and in malignant tumours, grade 1– 3 and dedifferentiated
Grade
n
Benign Grade 1 Grade 2 Grade 3 Dedifferentiated
12 12 8 4 2
PTHrP ( %) * 23.4 67.5 79.4 95.0 100.0
± ± ± ± ±
27.8 18.1 16.3 7.1 0.0
PTHrP receptor ( %) * 28.1 62.1 83.1 95.0 97.5
± ± ± ± ±
28.6 25.7 14.1 10.0 3.5
* PTHrP or PTH/ PTHrP receptor positivity is expressed as percentage of positive staining cells in a area of 1000 tumour cells in each of three random locations on one representative section.
grade tumour indicated that a similar factor could activate both expression of PTHrP and PTH/PTHrP receptor, since there was a strong correlation between the two in the same tumour. The differentiation between low grade chondrosarcom a and enchondroma can be difficult.3 This has an important impact on treatment modalities where benign tumours may be managed conservatively, while malignant lesions require surgical removal. In this series, the expression of both PTHrP and PTH/PTHrP receptor in chondrosarcoma was significantly higher than that in benign cartilaginous tumours, indicating that immunohistological staining of PTHrP and its receptor may be useful for differentiating between low grade chondrosarcoma and a benign enchondroma. For example, the mean positivity for grade 1 chondrosarcoma was three times higher than that of an enchondroma. In addition, a cut-off index of 45% PTHrP positivity in this study indicated a sensitivity of 92% (24 of 26) and a specificity of 92% ( 11 of 12), and a cut-off index of 70% PTH/PTHrP receptor positivity suggested a sensitivity of 88% (23 of 26) and a specificity of 92% (11 of 12). PTHrP and PTH/PTHrP receptor co-expression has been studied in several cancers,29–31 and it was reported that PTHrP could activate the growth of metastatic tumour cells in bone through interaction with the PTH/PTHrP receptor.32 Our results also suggest the possibility that PTHrP could activate the growth of chondrosarcoma cells in an autocrine/ paracrine way in bone tissue, in the same way that PTHrP activates the growth of metastatic cancer cells in bone. This is the first report of the co-expression of PTHrP and PTH/PTHrP receptor in chondrosarcoma. High expression of PTH/PTHrP receptor as well as PTHrP itself was detected in high grade chondrosarcoma, implicating these factors in the growth and progression of malignancy of chondrosarcoma. ACKNOWLEDGEMENTS We would like to thank Pat Smith, Elizabeth Williams and Toni Harris for their help with immunohistochemistry. Address for correspondence: Professor P. F. M. Choong, Department of Orthopaedics, St Vincent’s Hospital, Level 3 Daly Wing, 41 Victoria Parade, Fitzroy, Vic 3065, Australia. E-mail: [email protected]
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