Recent Developments

Recent Developments (updated June 2006)

1. Dual hypoxia markers.

Ljungkvist et al used dual markers to study the lifetime of hypoxic cells in rodent tumors and human tumor xenografts (1). Pimonidazole HCl (Hypoxyprobe) was administered at variable times before tumor harvest. A second hypoxic marker, CCI-103 (Hypoxyprobe-F6), was injected later at a fixed time before harvest. Hypoxic cell turnover was defined as the decrease in the extent of pimonidazole binding relative to that for CCI-103F binding as measured by immunofluorescence. The half-life of hypoxic cell turnover was found to be 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. In summary, differences in hypoxic cell turnover rates exist among tumor lines with half-lives ranging from 17-49 hrs.

Kleiter et al used dual markers to compare the efficacy of oral versus intravenous pimonidazole as a marker of hypoxia in canine tumors (2). Pimonidazole HCl was injected intravenously or ingested. Seven hours later CCI-103F was administered intravenously. Tumor biopsies were collected 26-30 hours later and the extent of pimonidazole binding compared to that for CCI-103F. An excellent correlation was observed between pimonidazole and CCI-103F binding (r2 = 0.97). On average, the extent of pimonidazole binding exceeded that for CCI-103F by a factor 1.2. However, excess pimonidazole binding occurred over a wide range among tumors and even within the same tumor (range 1.0 –1.65). Kleiter et al demonstrated a pH effect on binding that favored pimonidazole binding at higher pH and concluded that pimonidazole might be more efficient at detecting fluctuating hypoxia in solid tumors (2).

2. Clinical studies of oxygen regulated protein expression.

Studies of correlations between pimonidazole binding and the expression of hypoxia inducible, endogenous proteins continue. The current state of affairs is summarized in the Table. Areas of spatial co-localization are often seen but overall correlations, while in some cases statistically significant, are generally weak for squamous cell carcinomas. Possible exceptions include carbonic anhydrase IX and erythropoietin expression. The strongest correlations occur in carcinomas derived from simple epithelia (bladder and breast) indicating, perhaps, that a difference in biology exists between these tumors and squamous cell carcinomas.

Table. Correlations between pimonidazole binding and endogenous “hypoxia marker” protein expression.

Endogenous Marker	Corr. Coeff.	Cancer Site	Ref.
Carbonic anhydrase IX	No correlation	Liver mets; colorectal Ca	(3)
	No correlation	Cervix SCC	(4)
	No correlation	Colon AdenoCa	(5)
	0.27 (p<0.001)	Cervix SCC	(6)
	0.36 (p=0.02)	H&N SCC	(7)
	0.6	Cervix SCC	(8)
	0.74 (p<0.0001)	Bladder Ca	(9)
	0.75	Cervix SCC	(10)
EGFR	No correlation	Colon AdenoCa	(5)
EPO	0.6	Breast Ca	(11, 12)
	0.74 (p=0.001)	H&N SCC	(13)
EPOR	No correlation	H&N SCC	(13, 14)
	0.63 (p=0.0001)	Breast Ca	(12)
Glut-1	No correlation	Liver mets; colorectal Ca	(3)
	0.45 (p=0.003)	Cervix SCC	(4)
	0.82 (p=0.0001)	Bladder Ca	(9, 15)
HIF-1a	0.24 (p=0.51)	H&N SCC	(16, 17)
	0.34 (p=0.04)	Cervix SCC	(6)
Involucrin	No correlation	Cervix and H&N SCC	(18-20)
Metallothionein	No correlation	Cervix and H&N SCC	(18)
Thymidine phosphorylase	No correlation	Cervix SCC	(21)
VEGF	No correlation	Cervix and H&N SCC	(22)
	No correlation	Colon AdenoCa	(5)

3. New clinical applications.

In the past, pimonidazole has been used to study hypoxia in squamous cell carcinomas of the head and neck and carcinomas of the bladder and breast. Carnell et al have extended the use of pimonidazole to studies of hypoxia in prostate cancer (23). Working with biopsy material from 37 patients these investigators found that pimonidazole binding occurred in over 90% of prostate carcinoma and benign prostatic hyperplasias. A positive correlation between heavy pimonidazole binding and Gleason score was found (Spearman’s rank test, p = 0.044). A similar trend linking pimonidazole binding to differentiation has been reported for squamous cell carcinomas (19).

Goethals et al (5) and van Laarhoven et al (3) have extended the use of pimonidazole to include studies of colorectal adenocarcinoma. In neither study was a correlation between endogenous “hypoxic marker”expression and pimonidazole binding observed.

4. Normal tissue applications.

Applications of Hypoxyprobe to studies of normal tissue hypoxia continue to be reported. An updated list as of June 2006 categorized according to tissue type can be accessed via the Applications of Hypoxyprobe link.

5. Validation of non-invasive assays for hypoxia

A number of investigators have used Hypoxyprobe as a validation tool in the development of non-invasive assays of hypoxia including contrast enhanced magnetic resonance spectroscopy (24); positron emission tomography (25-30); and, urinary markers (31). Literature citations

1. Ljungkvist, A. S., Bussink, J., Kaanders, J. H., Rijken, P. F., Begg, A. C., Raleigh, J. A., and van der Kogel, A. J. Hypoxic cell turnover in different solid tumor lines. Int J Radiat Oncol Biol Phys, 62: 1157-1168, 2005.

2. Kleiter, M. M., Thrall, D. E., Malarkey, D. E., Ji, X., Lee, D. Y., Chou, S. C., and Raleigh, J. A. A comparison of oral and intravenous pimonidazole in canine tumors using intravenous CCI-103F as a control hypoxia marker. Int J Radiat Oncol Biol Phys, 64: 592-602, 2006.

3. van Laarhoven, H. W., Kaanders, J. H., Lok, J., Peeters, W. J., Rijken, P. F., Wiering, B., Ruers, T. J., Punt, C. J., Heerschap, A., and van der Kogel, A. J. Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer. Int J Radiat Oncol Biol Phys, 64: 473-482, 2006.

4. Airley, R. E., Loncaster, J., Raleigh, J. A., Harris, A. L., Davidson, S. E., Hunter, R. D., West, C. M., and Stratford, I. J. GLUT-1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: Relationship to pimonidazole binding. Int. J. Cancer, 104: 85-91, 2003.

5. Goethals, L., Debucquoy, A., Perneel, C., Geboes, K., Ectors, N., De Schutter, H., Penninckx, F., McBride, W. H., Begg, A. C., and Haustermans, K. M. Hypoxia in human colorectal adenocarcinoma: Comparison between extrinsic and potential intrinsic hypoxia markers. Int J Radiat Oncol Biol Phys, 65: 246-254, 2006.

6. Hutchison, G. J., Valentine, H. R., Loncaster, J. A., Davidson, S. E., Hunter, R. D., Roberts, S. A., Harris, A. L., Stratford, I. J., Price, P. M., and West, C. M. Hypoxia-inducible factor 1alpha expression as an intrinsic marker of hypoxia: correlation with tumor oxygen, pimonidazole measurements, and outcome in locally advanced carcinoma of the cervix. Clin Cancer Res, 10: 8405-8412, 2004.

7. Kaanders, J. H., Wijffels, K. I., Marres, H. A., Ljungkvist, A. S., Pop, L. A., van den Hoogen, F. J., de Wilde, P. C., Bussink, J., Raleigh, J. A., and van der Kogel, A. J. Pimonidazole binding and tumor vascularity predict for treatment outcome in head and neck cancer. Cancer Res., 62: 7066-7074, 2002.

8. Jankovic, B., Aquino-Parsons, C., Raleigh, J. A., Stanbridge, E. J., Durand, R. E., Banath, J. P., MacPhail, S. H., and Olive, P. L. Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix. Cytometry B Clin Cytom, 70: 45-55, 2006.

9. Hoskin, P. J., Sibtain, A., Daley, F. M., and Wilson, G. D. GLUT1 and CAIX as intrinsic markers of hypoxia in bladder cancer: relationship with vascularity and proliferation as predictors of outcome of ARCON. Br J Cancer, 89: 1290-1297, 2003.

10. Olive, P. L., Aquino-Parsons, C., MacPhail, S. H., Liao, S. Y., Raleigh, J. A., Lerman, M. I., and Stanbridge, E. J. Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer. Cancer Res, 61: 8924-8929, 2001.

11. Arcasoy, M. O., Amin, K., Karayal, A. F., Chou, S. C., Raleigh, J. A., Varia, M. A., and Haroon, Z. A. Functional significance of erythropoietin receptor expression in breast cancer. Lab Invest, 82: 911-918, 2002.

12. Raleigh, J. A., Arcasoy, M. O., Amikn, K., Lininger, R. A., and Varia, M. A. Comparison between exogenous and endogenous hypoxia markers in human cancer: pimonidazole and erythropoietin. . In: American Association for Cancer Research 97th Annual Meeting, Washington, DC2006, pp. Abstract #3631.

13. Arcasoy, M. O., Amin, K., Chou, S. C., Haroon, Z. A., Varia, M., and Raleigh, J. A. Erythropoietin and erythropoietin receptor expression in head and neck cancer: relationship to tumor hypoxia. Clin Cancer Res, 11: 20-27, 2005.

14. Hoogsteen, I. J., Peeters, W. J., Marres, H. A., Rijken, P. F., van den Hoogen, F. J., van der Kogel, A. J., and Kaanders, J. H. Erythropoietin receptor is not a surrogate marker for tumor hypoxia and does not correlate with survival in head and neck squamous cell carcinomas. Radiother Oncol, 76: 213-218, 2005.

15. Wykoff, C. C., Beasley, N. J., Watson, P. H., Turner, K. J., Pastorek, J., Sibtain, A., Wilson, G. D., Turley, H., Talks, K. L., Maxwell, P. H., Pugh, C. W., Ratcliffe, P. J., and Harris, A. L. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res, 60: 7075-7083, 2000.

16. Janssen, H. L., Haustermans, K. M., Sprong, D., Blommestijn, G., Hofland, I., Hoebers, F. J., Blijweert, E., Raleigh, J. A., Semenza, G. L., Varia, M. A., Balm, A. J., van Velthuysen, M. L., Delaere, P., Sciot, R., and Begg, A. C. HIF-1alpha, pimonidazole, and iododeoxyuridine to estimate hypoxia and perfusion in human head-and-neck tumors. Int J Radiat Oncol Biol Phys, 54: 1537-1549, 2002.

17. Begg, A. C. Is HIF-1alpha a good marker for tumor hypoxia? Int. J. Radiat. Oncol. Biol. Phys., 56: 917-919, 2003.

18. Raleigh, J. A., Chou, S. C., Calkins-Adams, D. P., Ballenger, C. A., Novotny, D. B., and Varia, M. A. A clinical study of hypoxia and metallothionein protein expression in squamous cell carcinomas. Clin Cancer Res, 6: 855-862, 2000.

19. Azuma, Y., Chou, S. C., Lininger, R. A., Murphy, B. J., Varia, M. A., and Raleigh, J. A. Hypoxia and differentiation in squamous cell carcinomas of the uterine cervix: pimonidazole and involucrin. Clin Cancer Res, 9: 4944-4452, 2003.

20. Chou, S. C., Azuma, Y., Varia, M. A., and Raleigh, J. A. Evidence that involucrin, a marker for differentiation, is oxygen regulated in human squamous cell carcinomas. Br. J. Cancer, 90: 728-735, 2004.

21. Kabuubi, P., Loncaster, J. A., Davidson, S. E., Hunter, R. D., Kobylecki, C., Stratford, I. J., and West, C. M. No relationship between thymidine phosphorylase (TP, PD-ECGF) expression and hypoxia in carcinoma of the cervix. Br J Cancer, 94: 115-120, 2006.

22. Raleigh, J. A., Calkins-Adams, D. P., Rinker, L. H., Ballenger, C. A., Weissler, M. C., Fowler, W. C., Jr., Novotny, D. B., and Varia, M. A. Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res, 58: 3765-3768, 1998.

23. Carnell, D. M., Smith, R. E., Daley, F. M., Saunders, M. I., Bentzen, S. M., and Hoskin, P. J. An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: Implications for radioresistance. Int J Radiat Oncol Biol Phys, 65: 91-99, 2006.

24. Ceelen, W., Smeets, P., Backes, W., Van Damme, N., Boterberg, T., Demetter, P., Bouckenooghe, I., De Visschere, M., Peeters, M., and Pattyn, P. Noninvasive monitoring of radiotherapy-induced microvascular changes using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in a colorectal tumor model. Int J Radiat Oncol Biol Phys, 64: 1188-1196, 2006.

25. Dearling, J. L., Flynn, A. A., Sutcliffe-Goulden, J., Petrie, I. A., Boden, R., Green, A. J., Boxer, G. M., Begent, R. H., and Pedley, R. B. Analysis of the regional uptake of radiolabeled deoxyglucose analogs in human tumor xenografts. J. Nucl. Med., 45: 101-107, 2004.

26. Dubois, L., Landuyt, W., Haustermans, K., Dupont, P., Bormans, G., Vermaelen, P., Flamen, P., Verbeken, E., and Mortelmans, L. Evaluation of hypoxia in an experimental rat tumour model by [(18)F]fluoromisonidazole PET and immunohistochemistry. Br J Cancer, 91: 1947-1954, 2004.

27. O'Donoghue J, A., Zanzonico, P., Pugachev, A., Wen, B., Smith-Jones, P., Cai, S., Burnazi, E., Finn, R. D., Burgman, P., Ruan, S., Lewis, J. S., Welch, M. J., Ling, C. C., and Humm, J. L. Assessment of regional tumor hypoxia using (18)F-fluoromisonidazole and (64)Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) positron emission tomography: Comparative study featuring microPET imaging, Po(2) probe measurement, autoradiography, and fluorescent microscopy in the R3327-AT and FaDu rat tumor models. Int J Radiat Oncol Biol Phys, 61: 1493-1502, 2005.

28. Pugachev, A., Ruan, S., Carlin, S., Larson, S. M., Campa, J., Ling, C. C., and Humm, J. L. Dependence of FDG uptake on tumor microenvironment. Int J Radiat Oncol Biol Phys, 62: 545-553, 2005.

29. Dorow, D. S., Cullinane, C., Conus, N., Roselt, P., Binns, D., McCarthy, T. J., McArthur, G. A., and Hicks, R. J. Multi-tracer small animal PET imaging of the tumour response to the novel pan-Erb-B inhibitor CI-1033. Eur J Nucl Med Mol Imaging: 1-12, 2006.

30. Yuan, H., Schroeder, T., Bowsher, J. E., Hedlund, L. W., Wong, T., and Dewhirst, M. W. Intertumoral differences in hypoxia selectivity of the PET imaging agent 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone). J Nucl Med, 47: 989-998, 2006.

31. Nelson, D. W., Cao, H., Zhu, Y., Sunar-Reeder, B., Choi, C. Y., Faix, J. D., Brown, J. M., Koong, A. C., Giaccia, A. J., and Le, Q. T. A noninvasive approach for assessing tumor hypoxia in xenografts: developing a urinary marker for hypoxia. Cancer Res, 65: 6151-6158, 2005.