Hypoxia Markers in Human Osteosarcoma: An Exploratory Study

  • Hiroo Mizobuchi
  • José Manuel García-Castellano
  • Shaji Philip
  • John H. Healey
  • Richard Gorlick
Symposium: Molecular Genetics in Sarcoma


Neoplastic cells growing under hypoxic conditions exhibit a more aggressive phenotype by activating a cascade of molecular events partly mediated by hypoxia-inducible transcription factor (HIF-1α) and vascular endothelial growth factor (VEGF). The roles of these markers have been studied previously in several cancer lines. We ascertained the frequency of HIF-1α expression, VEGF expression, the degree of neovascularization, and cell proliferation in osteosarcoma samples. Samples from osteosarcoma patients were assessed for HIF-1α and VEGF protein expression using immunohistochemistry, neovascularization using antibodies for Factor VIII, and cell proliferation using the Ki-67 labeling index. Associations between these parameters and clinical features were examined. HIF-1α staining was positive in 35% of patients and metastases were present in 61% of these HIF-1α-positive patients. VEGF protein expression was detected in 69% of patients, 92% of whom were female. We observed an insignificant trend for a higher frequency of VEGF expression in the high-grade as compared to low-grade osteosarcoma. We observed no association between vascular density and proliferation index and any clinical parameters. We found an association between HIF-1α expression and metastatic disease and between VEGF expression and female gender.


  1. 1.
    Bolger BS, Symonds RP, Stanton PD, MacLean AB, Burnett R, Kelly P, Cooke TG. Prediction of radiotherapy response of cervical carcinoma through measurement of proliferation rate. Br J Cancer. 1996;74:1223–1226.PubMedGoogle Scholar
  2. 2.
    Bos R, van Diest PJ, van der Groep P, Shvarts A, Greijer AE, van der Wall E. Expression of hypoxia-inducible factor-1alpha and cell cycle proteins in invasive breast cancer are estrogen receptor related. Breast Cancer Res. 2004;6:R450–R459.PubMedCrossRefGoogle Scholar
  3. 3.
    Chau NM, Rogers P, Aherne W, Carroll V, Collins I, McDonald E, Workman P, Ashcroft M. Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1α induction in response to hypoxic stress and growth factors. Cancer Res. 2005;65:4918–4928.PubMedCrossRefGoogle Scholar
  4. 4.
    Chen WT, Huang CJ, Wu MT, Yang SF, Su YC, Chai CY. Hypoxia-inducible factor-1alpha is associated with risk of aggressive behavior and tumor angiogenesis in gastrointestinal stromal tumor. Jpn J Clin Oncol. 2005;35:207–213.PubMedCrossRefGoogle Scholar
  5. 5.
    Cole DJ, Brown DC, Crossley E, Alcock CJ, Gatter KC. Carcinoma of the cervix uteri: an assessment of the relationship of tumour proliferation to prognosis. Br J Cancer. 1992;65:783–785.PubMedGoogle Scholar
  6. 6.
    Eggert A, Ikegaki N, Kwiatkowski J, Zhao H, Brodeur GM, Himelstein BP. High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas. Clin Cancer Res. 2000;6:1900–1908.PubMedGoogle Scholar
  7. 7.
    Ferrandina G, Ranelletti FO, Gallotta V, Martinelli E, Zannoni GF, Gessi M, Scambia G. Expression of cyclooxygenase-2 (COX-2), receptors for estrogen (ER), and progesterone (PR), p53, ki67, and neu protein in endometrial cancer. Gynecol Oncol. 2005;98:383–389.PubMedCrossRefGoogle Scholar
  8. 8.
    Ferrara N. VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer. 2002;2:795–803.PubMedCrossRefGoogle Scholar
  9. 9.
    Fyles AW, Milosevic M, Wong R, Kavanagh MC, Pintilie M, Sun A, Chapman W, Levin W, Manchul L, Keane TJ, Hill RP. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol. 1998;48:149–156.PubMedCrossRefGoogle Scholar
  10. 10.
    Giatromanolaki A, Koukourakis MI, Kakolyris S, Turley H, O’Byrne K, Scott PA, Pezzella F, Georgoulias V, Harris AL, Gatter KC. Vascular endothelial growth factor, wild-type p53, and angiogenesis in early operable non-small lung cancer. Clin Cancer Res. 1997;3:2485–2492.PubMedGoogle Scholar
  11. 11.
    Graeber TG, Osmanian C, Jacks T, Housman DE, Kock CJ, Lowe SW, Giaccia AJ. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature. 1996;379:88–91.PubMedCrossRefGoogle Scholar
  12. 12.
    Grau C, Overgaard J. Effect of etoposide, carmustine, vincristine, 5–fluorouracil, or methotrexate on radiobiologically oxic and hypoxic cells in a C3H mouse mammary carcinoma in situ. Cancer Chemother Pharmacol. 1992;30:277–280.PubMedCrossRefGoogle Scholar
  13. 13.
    Hockel M, Knoop C, Schlenger K, Vorndran B, Baussmann E, Mitze M, Knapstein PG, Vaupel P. Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix. Radiother Oncol. 1993;26:45–50.PubMedCrossRefGoogle Scholar
  14. 14.
    Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res. 1996;56:4509–4515.PubMedGoogle Scholar
  15. 15.
    Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst. 2001;93:266–276.PubMedCrossRefGoogle Scholar
  16. 16.
    Iyer NV, Leung SW, Semenza GL. The human hypoxia-inducible factor 1α gene: HIF1A structure and evolutionary conservation. Genomics. 1998;52:159–165.PubMedCrossRefGoogle Scholar
  17. 17.
    Junior AT, de Abreu Alves F, Pinto CA, Carvalho AL, Kowalski LP, Lopes MA. Clinicopathological and immunohistochemical analysis of twenty-five head and neck osteosarcomas. Oral Oncol. 2003;39:521–530.PubMedCrossRefGoogle Scholar
  18. 18.
    Kaya M, Wada T, Akatsuka T, Kawaguchi S, Nagoya S, Shindoh M, Higashino F, Mezawa F, Okada F, Ishii S. Vascular endothelial growth factor expression in untreated osteosarcoma is predictive of pulmonary metastasis and poor prognosis. Clin Cancer Res. 2000;6:572–577.PubMedGoogle Scholar
  19. 19.
    Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science. 1989;246:1309–1312.PubMedCrossRefGoogle Scholar
  20. 20.
    Koong AC, Denko NC, Hudson KM, Schindler C, Swiersz L, Koch C, Evans S, Ibrahim H, Le QT, Terris DJ, Giaccia AJ. Candidate genes for the hypoxic tumor phenotype. Cancer Res. 2000;60:883–887.PubMedGoogle Scholar
  21. 21.
    Koos RD, Kazi AA, Roberson MS, Jones JM. New insight into the transcriptional regulation of vascular endothelial growth factor expression in the endometrium by estrogen and relaxin. Ann N Y Acad Sci. 2005;1041:233–247.PubMedCrossRefGoogle Scholar
  22. 22.
    Lagrange JL, Courdi A, Chauvel P, Gioanni J, Ettore F, Bongain A, Duforestel T, Gillet JY. The labelling index in carcinoma of the uterine cervix: its correlation with tumour sterilization. Br J Radiol. 1992;65:63–65.PubMedCrossRefGoogle Scholar
  23. 23.
    Lee AH, Dublin EA, Bobrow LG, Poulsom R. Invasive lobular and invasive ductal carcinoma of the breast show distinct patterns of vascular endothelial growth factor expression and angiogenesis. J Pathol. 1998;185:394–401.PubMedCrossRefGoogle Scholar
  24. 24.
    Lee YH, Tokunaga T, Oshika Y, Suto R, Yanagisawa K, Tomisawa M, Fukuda H, Nakano H, Abe S, Tateishi A, Kijima H, Yamazaki H, Tamaoki N, Ueyama Y, Nakamura M. Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma. Eur J Cancer. 1999;35:1089–1093.PubMedCrossRefGoogle Scholar
  25. 25.
    Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev. 2000;21:115–137.PubMedCrossRefGoogle Scholar
  26. 26.
    Manolagas SC, Kousteni S, Jilka RL. Sex steroids and bone. Recent Prog Horm Res. 2002;57:385–409.PubMedCrossRefGoogle Scholar
  27. 27.
    Mueller MD, Vigne JL, Minchenko A, Lebovic DI, Leitman DC, Taylor RN. Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta. Proc Natl Acad Sci USA. 2000;97:10972–10977.PubMedCrossRefGoogle Scholar
  28. 28.
    Nakano T, Oka K, Arai T. Histological and immunohistochemical prediction for local control of cervical squamous cell carcinoma treated with radiotherapy alone. Int J Radiation Oncology Biol Phys. 1990;19:1011–1019.Google Scholar
  29. 29.
    Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, Prusiner SB. Conversion of alpha-helices beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA. 1993;90:10962–10966.PubMedCrossRefGoogle Scholar
  30. 30.
    Peng TS, Qiu JS, Wu HX, Liang HZ, Luo CQ. Expressions of CD44s, MMP-9, and Ki-67: possible association with invasion, metastasis, and recurrence of osteosarcoma. Ai Zheng. 2002;21:745–750.PubMedGoogle Scholar
  31. 31.
    Perrone G, Santini D, Vincenzi B, Zagami M, La Cesa A, Bianchi A, Altomare V, Primavera A, Battista C, Vetrani A, Tonini G, Rabitti C. COX-2 expression in DCIS: correlation with VEGF, HER-2/neu, prognostic molecular markers and clinicopathological features. Histopathology. 2005;46:561–568.PubMedCrossRefGoogle Scholar
  32. 32.
    Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol. 2001;19:1207–1225.PubMedGoogle Scholar
  33. 33.
    Riggs BL, Khosla S, Melton LJ 3rd. A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res. 1998;13:763–773.PubMedCrossRefGoogle Scholar
  34. 34.
    Riggs BL, Khosla S, Melton LJ 3rd. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev. 2002;23:279–302.PubMedCrossRefGoogle Scholar
  35. 35.
    Rofstad EK, Danielsen T. Hypoxia-induced metastasis of human melanoma cells: involvement of vascular endothelial growth factor-mediated angiogenesis. Br J Cancer. 1999;80:169–707.CrossRefGoogle Scholar
  36. 36.
    Rosen G, Caparros B, Huvos AG, et al. Preoperative chemotherapy for osteogenic sarcoma: Selection of postoperative adjuvant chemotherapy based on the response of the primary tumor to preoperative chemotherapy. Cancer 1982;49:1221–1230.PubMedCrossRefGoogle Scholar
  37. 37.
    Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999;15:551–578.PubMedCrossRefGoogle Scholar
  38. 38.
    Subbaramaiah K, Dannenberg AJ. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol Sci. 2003;24:96–102.PubMedCrossRefGoogle Scholar
  39. 39.
    Tan C, de Noronha RG, Roecker AJ, Pyrzynska B, Khwaja F, Zhang Z, Zhang H, Teng Q, Nicholson AC, Giannakakou P, Zhou W, Olson JJ, Pereira MM, Nicolaou KC, Van Meir EG. Identification of a novel small-molecule inhibitor of the hypoxia-inducible factor 1 pathway. Cancer Res. 2005; 65:605–612.PubMedGoogle Scholar
  40. 40.
    Tsang RW, Fyles AW, Kirkbride P, Levin W, Manchul LA, Milosevic MF, Rawlings GA, Banerjee D, Pintilie M, Wilson GD. Proliferation measurements with flow cytometry Tpot in cancer of the uterine cervix: correlation between two laboratories and preliminary clinical results. Int J Radiat Oncol Biol Phys. 1995;32:1319–1329.PubMedGoogle Scholar
  41. 41.
    Tsang RW, Fyles AW, Milosevic M, Syed A, Pintilie M, Levin W, Manchul LA. Interrelationship of proliferation and hypoxia in carcinoma of the cervix. Int J Radiation Oncology Biol Phys. 2000;46:95–99.CrossRefGoogle Scholar
  42. 42.
    Tsang RW, Wong CS, Fyles AW, Levin W, Manchul LA, Milosevic M, Chapman W, Li YQ, Pintilie M. Tumour proliferation, apoptosis in human uterine cervix carcinoma II: correlations with clinical outcome. Radiother Oncol. 1999;50:93–101.PubMedCrossRefGoogle Scholar
  43. 43.
    Vaupel P, Kallinowski F, Okuniell P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 1989;49:6449–6465.PubMedGoogle Scholar
  44. 44.
    Vaupel P, Mayer A. Hypoxia in cancer: Significance and impact on clinical outcome. Cancer Metastasis Rev. 2007;26:225–239.PubMedCrossRefGoogle Scholar
  45. 45.
    Willet CG, Warland G, Hagan MP, Daly WJ, Coen J, Shellito PC, Compton CC. Tumor proliferation in rectal cancer following preoperative irradiation. J Clin Oncol. 1995;13:1417–1424.Google Scholar
  46. 46.
    Wouters BG, Brown JM. Cells at intermediate oxygen levels can be more important than the “hypoxic fraction” in determining tumor response to fractionated radiotherapy. Radiat Res. 1997;147:541–550.PubMedCrossRefGoogle Scholar
  47. 47.
    Wu Q, Yang SH, Wang RY, Ye SN, Xia T, Ma DZ. Effect of silencing HIF-1alpha by RNA interference on expression of vascular endothelial growth factor in osteosarcoma cell line SaOS-2 under hypoxia. Ai Zheng. 2005;24:531–535.PubMedGoogle Scholar
  48. 48.
    Wu Q, Yang SH, Ye SN, Wang RY. Therapeutic effects of RNA interference targeting HIF-1 alpha gene on human osteosarcoma. Zhonghua Yi Xue Za Zhi. 2005;85:409–413.PubMedGoogle Scholar
  49. 49.
    Zheng MH, Xu J, Robbins P, Pavlos N, Wysocki S, Kumta SM, Wood DJ, Papadimitriou JM. Gene expression of vascular endothelial growth factor in giant cell tumors of bone. Hum Pathol 2000;31:804–812.PubMedCrossRefGoogle Scholar
  50. 50.
    Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, Buechler P, Isaacs WB, Semenza GL, Simons JW. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res. 1999;59:5830–5835.PubMedGoogle Scholar
  51. 51.
    Zhu ZH, Rong TH, Zeng CG, Wu QL, Ma Y, Huang XP, Li BJ, Zhang PY, Zhao JM, Hu W, Zhang SY, Yu H, Ma GW, Zhang LJ, Wen ZS, Fu JH, Long H. Vascular endothelial growth factor expression and microvessel density in Stage I–II non-small cell lung cancer and their prognostic significances. Ai Zheng. 2005;24:865–869.PubMedGoogle Scholar

Copyright information

© The Association of Bone and Joint Surgeons 2008

Authors and Affiliations

  • Hiroo Mizobuchi
    • 1
  • José Manuel García-Castellano
    • 1
  • Shaji Philip
    • 2
  • John H. Healey
    • 1
  • Richard Gorlick
    • 2
  1. 1.Department of SurgeryMemorial Sloan-Kettering Cancer CenterNew YorkUSA
  2. 2.Division of Hematology/Oncology, Department of PediatricsThe Children’s Hospital at MontefioreBronxUSA

Personalised recommendations