Postgraduate Medical Institute of the University of Hull in association with Hull York Medical School, University of Hull, Hull, UK
Received 3 December 2003; revised 20 January 2004; accepted 21 January 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Radiotherapy is the principal modality used to treat early stage laryngeal cancer. Unfortunately treatment failures occur in 1025% of patients. Subsequent salvage surgery is technically more difficult, with increased complication and failure rates. The ability to predict or prevent radioresistance would improve the poor survival associated with this disease. Cox-2 is an inducible enzyme involved with prostaglandin synthesis. We investigated a potential role for Cox-2 in predicting radioresistance in laryngeal cancer.
Patients and methods:
Using immunohistochemical techniques we examined the expression of Cox-2 protein in 122 pre-treatment laryngeal biopsies. All tumours were treated with single modality radiotherapy (curative intent). The group comprised of 61 radioresistant and 61 radiosensitive tumours matched for T stage, laryngeal subsite, gender and smoking history.
Results:
Cox-2 expression was detected in 41 of 61 (67%) biopsy samples from patients with radioresistant tumours and 25 of 61 (41%) radiosensitive tumours. Overexpression was significantly associated with radioresistant tumours (P = 0.004). Cox-2 has a 67% accuracy in predicting radiotherapy failure.
Conclusion:
Cox-2 may have prognostic value in predicting response to radiotherapy. Cox-2 inhibitors such as NS-398 have been shown to enhance the effects of radiotherapy. We suggest that their use may be beneficial in patients who are destined to fail radiotherapy.
Key words: Cox-2; laryngeal cancer; radioresistance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Radiotherapy used as a single treatment modality can be an effective cure for early stage (T1 and T2) laryngeal tumours. Radiotherapy treatment has the advantage over a total laryngectomy in that it spares functional anatomy and the patient is able to speak and swallow normally following therapy. The loss of such abilities has a significant impact on the quality of a patients life. The subsequent impaired ability to communicate and the disfiguring surgery also have a detrimental psychological impact. Unfortunately radiotherapy treatment failures do occur. Approximately 10% of patients with stage I disease [5] and 25% of patients with stage II disease [6] do not respond to radiotherapy. These observations show that the TNM (tumournodemetastasis) system, although widely used as the basis for patient cancer management, cannot however predict an individual tumour response to radiotherapy [7].
If a patient fails radiotherapy, a total laryngectomy is the main treatment option that may offer a cure. The loss of the larynx has a significant psychological impact upon the patient, especially one who has undergone a failed course of radiotherapy. Operating in a previously irradiated field results in increased surgical failure and complication rates [8]. More importantly, definitive cancer cure is delayed by the course of radiotherapy. Due to this delay tumour progression may have occurred, adversely affecting patient prognosis still further.
The prostaglandin system is composed of two distinct isoenzymes: cyclooxygenase 1 and 2. These enzymes form part of the prostaglandin synthetase complex of enzymes, which play a key role in the conversion of archidonic acid into prostaglandin G2 and H2. Prostaglandin H2 is then transformed into individual prostaglandins (PGD2, PGE2, PGF2, PGI2, TXA2) by tissue-specific components of the synthetase complex. These individual components can then exert their effects on angiogenesis, apoptosis and immune surveillance. Recently, Cox-2 protein over-expression has been reported to correlate with decreased survival in patients with cervical cancer after treatment with radiotherapy [9]. Cox-2 is an inducible enzyme not normally present in normal tissue in contrast to Cox-1, which is expressed at a constant level throughout the cell cycle by almost all tissues [10]. Cox-2 can be induced by a variety of stimuli including cytokines [11], growth factors [12] and oncogenes [10]. Prostaglandins regulated by Cox-2 are believed to be important in the pathogenesis of cancer due to their effects on apoptosis, angiogenesis, cell proliferation and immune surveillance [10, 13]. The precise mechanistic role for Cox-2 in tumorigenesis continues to be evaluated; however, its overexpression is believed to play an important role [10]. In support of this, Oshim et al. have demonstrated that knocking out the Cox-2 gene leads to a marked reduction in intestinal polyps, in a mouse model of familial adenomatous polyposis [14].
Cox-2 overexpression has also been implicated in tumour response to radiotherapy. Tsujii and Dubois demonstrated that cell lines which overexpress Cox-2 were resistant to apoptosis, an important pathway of cell death induced by ionising radiation [15]. Pyo et al. demonstrated that the Cox-2 inhibitor, NS-398, enhances the effect of radiotherapy in vitro and in vivo on human cells that overexpress Cox-2 [16]. These authors reported that the radiation-enhancing effects of NS-398 did not occur in cells deficient in Cox-2 expression and concluded that Cox-2 over-expression was essential for the effects of NS-398. On the basis of these observations we investigated the possible relationship between Cox-2 protein expression and treatment failure in laryngeal cancer treated with radiotherapy.
![]() |
Patients and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The radioresistant group consisted of 61 patients: 43 stage T1 and 18 stage T2 laryngeal squamous cell carcinomas. The criteria for a radioresistant tumour were: (a) the radiotherapy had to be given as a single modality treatment with curative intent for a biopsy-proven squamous cell carcinoma of the larynx; (b) biopsy-proven recurrent squamous cell carcinoma, the recurrence occurring at the original anatomical site, within 12 months of finishing a course of radiotherapy.
The radiosensitive group of tumours consisted of 61 patients: 43 stage T1 and 18 stage T2 squamous cell carcinomas of the larynx. The criteria for a radiosensitive tumour were: (a) the radiotherapy had to be given as a single modality treatment with curative intent for a biopsy-proven squamous cell carcinoma of the larynx; (b) post treatment, patients had a minimum follow-up of 3 years with no evidence of a recurrent laryngeal tumour.
Tissue sections (4 µm) were cut from pre-treatment archival tissue blocks of the radioresistant and radiosensitive tumours. Immunohistochemistry as previously described was used to detect Cox-2 on the tissue sections [18]. In brief, antigen retrieval was performed using pressurised heat retrieval. The primary antibody (100 µl), anti-Cox-2 (BD Biosciences, USA; Cat no. 610203 clone 33), at a dilution of 1:50 (diluted in 0.2x casein) was added to each tissue section and incubated at room temperature for 2 h. A negative control was included using 100 µl of 0.2x casein instead of the primary antibody. The Duet kit (DAKO, Denmark) was used as the secondary detection system and 3,3'-diaminobenzidine tetrachloride as the chromogen. Two assessors scored the anti-Cox-2 staining independently with the radiotherapy treatment outcome blinded to the assessor. A simple scoring system analysing only tumour cells was used to interpret the staining patterns [19]. Sections were regarded as positive if >5% of the tumour cells stained. Sections with 5% of the tumour staining were considered negative. In order to reduce sampling error the whole biopsy section for each tumour was analysed.
Statistics
Chi-squared statistical analysis using SPSS version 11.5 (SPSS Inc, Chicago, USA) was used throughout. All P values quoted are for two-sided significance, between the radioresistant and radiosensitive groups. Values <0.05 were considered significant. Marker sensitivity and specificity were calculated as previously described [20].
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
If Cox-2 is used as a predictive marker for radiotherapy outcome in early stage laryngeal cancer it has an accuracy of 67% (Table 2).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Head and neck cancers comprise a heterogeneous group of tumours arising from the upper aerodigestive tract, paranasal sinuses and glandular tissue. Each region has its own TNM staging system and differing response rates to radiotherapy [3]. We have limited this series to the larynx, which forms the largest subgroup of head and neck cancers. This is again to reduce confounding factors that may affect results from mixed tumour groups, as it is well documented that for a given T stage regional variation for treatment modality and outcome exists [3].
Using only pre-treatment tissue we have demonstrated that Cox-2 is overexpressed in laryngeal cancer. This is in agreement with previous observations of Cox-2 protein expression in head and neck cancer. Chan et al. reported that Cox-2 is expressed in 100% of tumours from a mixed group of 10 head and neck patients [24]. Ranelletti et al. reported Cox-2 overexpression in 31% (19 of 61) of tumours that comprised 18 stage I or II laryngeal cancers and 43 stage III or IV laryngeal cancers [25].
Our series represent a large collection of radioresistant tumours from one head and neck sub-site. It is the first study to document the overexpression of Cox-2 in such a group. We have found that 67% of radioresistant tumours overexpress Cox-2 in pre-treatment biopsy samples. The radioresistant tumour group have also been matched for T stage, laryngeal subsite and smoking history to a group of radiosensitive tumours. We have shown that Cox-2 overexpression has a greater association with radioresistant tumours than radiosensitive tumours (P = 0.004).
The association of Cox-2 with the radioresistant tumours has two potentially important consequences. First, as this association is present in pre-treatment biopsy material it may be used as a prognostic marker predicting radiotherapy treatment failure with an accuracy of 67%. Cox-2-positive patients with early T1 or T2 laryngeal cancer could be offered partial laryngeal surgery as a first-line treatment instead of radiotherapy. This treatment option is widely used in the USA and is equally as effective as radiotherapy for early stage laryngeal tumours [3]. Consequently such patients will not require salvage surgery and will benefit from improved survival and quality of life as their larynx will be preserved and they will not have to receive unnecessary radiotherapy. Equally there will be no detrimental effect to the 20% of patients with a false positive Cox-2 result who will be offered partial laryngeal surgery instead of radiotherapy. The accuracy of prediction may be increased if further markers can be identified which could be used in combination with Cox-2.
Second, the use of Cox-2 inhibitors may be able to enhance the effects of radiotherapy. Cells overexpressing Cox-2 have been shown to be resistant to apoptosis, an important mechanism of radiation-induced cell death [15]. The authors attributed this effect to increased levels of the important anti-apoptotic protein bcl-2, in epithelial cells, with forced overexpression of Cox-2. In the clinical setting, overexpression of Cox-2 has been associated with radiotherapy treatment failures in cervical cancer [9, 26, 27].
Inhibitors of Cox-2 have been shown to induce apoptosis in tumour cells [28, 29]. The Cox-2 inhibitor, NS-398, enhances the effect of radiotherapy, in vitro and in vivo, preferentially on human cell lines that overexpress Cox-2 [16]. Selective Cox-2 inhibitors, NS-398 and SC-236 [30], are non-toxic analgesic agents, which do not have the common gastrointestinal side of the commonly used non-selective Cox inhibitors such as aspirin [31]. It has also been shown that Cox-2 inhibitors do not affect the radiotherapy response of normal tissue in mice [32]. This is probably due to the lack of Cox-2 expression in normal tissue. This would allow the therapeutic ratio to be increased if Cox-2 inhibitors were used as a radiosensitiser for human cancers. By using Cox-2 inhibitors the clinician may be able to radiosensitise laryngeal cancer. This would result in fewer radiotherapy treatment failures. It would also spare the patient the functional loss and psychological impact associated with salvage surgery following treatment failure. Cox-2 inhibitors may also be beneficial to patients with advanced stage laryngeal cancer, who do not have the alternative treatment options that are available for early stage tumours. Advanced tumours are generally treated with combined surgery and radiotherapy in contrast to single modality treatment of early stage tumours [3].
Despite advances in radiotherapy treatment for laryngeal cancer, patient survival has not improved significantly over the last two decades [2]. The ability to predict radioresistant tumours would allow the clinician to use alternative therapies, aimed at achieving tumour control. Understanding the mechanism of tumour radioresistance will allow innovative therapies to be devised. Inhibitors of Cox-2 have the ability to enhance the effects of radiation. By using such inhibitors to radiosensitise tumours, enhanced radiotherapy cure rates may be attained. It is hoped that such strategies would improve the poor survival figures for head and neck cancer that have remained static since the 1980s.
![]() |
Acknowledgements |
---|
![]() |
FOOTNOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Parkin DM, Bray F, Ferlay J et al. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001; 94: 153156.[CrossRef][ISI][Medline]
3. Wilson JA. Effective Head and Neck Cancer Management, 3rd edition. London: British Association of Otorhinolaryngologists Head and Neck Surgeons 2002.
4. Cann CI, Fried MP, Rothman KJ. Epidemiology of squamous cell cancer of the head and neck. Otolaryngol Clin North Am 1985; 18: 367388.[ISI]
5. Klintenberg C, Lundgren J, Adell G et al. Primary radiotherapy of T1 and T2 glottic carcinomaanalysis of treatment results and prognostic factors in 223 patients. Acta Oncol 1996; 35 (Suppl 8): 8186.[ISI][Medline]
6. Fernberg JO, Ringborg U, Silfversward C et al. Radiation therapy in early glottic cancer. Analysis of 177 consecutive cases. Acta Otolaryngol 1989; 108: 478481.[ISI][Medline]
7. Smith BD, Haffty BG. Molecular markers as prognostic factors for local recurrence and radioresistance in head and neck squamous cell carcinoma. Radiat Oncol Investig 1999; 7: 125144.[CrossRef][ISI][Medline]
8. McLaughlin MP, Parsons JT, Fein DA et al. Salvage surgery after radiotherapy failure in T1T2 squamous cell carcinoma of the glottic larynx. Head Neck 1996; 18: 229235.[CrossRef][ISI][Medline]
9. Kim HJ, Wu HG, Park IA et al. High cyclooxygenase-2 expression is related with distant metastasis in cervical cancer treated with radiotherapy. Int J Radiat Oncol Biol Phys 2003; 55: 1620.[CrossRef][ISI][Medline]
10. Singh-Ranger G, Mokbel K. The role of cyclooxygenase-2 (COX-2) in breast cancer, and implications of COX-2 inhibition. Eur J Surg Oncol 2002; 28: 729737.[CrossRef][ISI][Medline]
11. Dempke W, Rie C, Grothey A et al. Cyclooxygenase-2: a novel target for cancer chemotherapy? J Cancer Res Clin Oncol 2001; 127: 411417.[CrossRef][ISI][Medline]
12. Fosslien E. Molecular pathology of cyclooxygenase-2 in neoplasia. Ann Clin Lab Sci 2000; 30: 321.[Abstract]
13. Lin DT, Subbaramaiah K, Shah JP et al. Cyclooxygenase-2: a novel molecular target for the prevention and treatment of head and neck cancer. Head Neck 2002; 24: 792799.[CrossRef][ISI][Medline]
14. Oshima M, Dinchuk JE, Kargman SL et al. Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 1996; 87: 803809.[ISI][Medline]
15. Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995; 83: 493501.[ISI][Medline]
16. Pyo H, Choy H, Amorino GP et al. A selective cyclooxygenase-2 inhibitor, NS-398, enhances the effect of radiation in vitro and in vivo preferentially on the cells that express cyclooxygenase-2. Clin Cancer Res 2001; 7: 29983005.
17. Greene FL, Sobin LH. The TNM system: our language for cancer care. J Surg Oncol 2002; 80: 119120.[CrossRef][ISI][Medline]
18. Cawkwell L, Gray S, Murgatroyd H et al. Choice of management strategy for colorectal cancer based on a diagnostic immunohistochemical test for defective mismatch repair. Gut 1999; 45: 409415.
19. Mighell AJ, Hume WJ, Robinson PA. An overview of the complexities and subtleties of immunohistochemistry. Oral Dis 1998; 4: 217223.[Medline]
20. Greenhalgh T. How to read a paper. Papers that report diagnostic or screening tests. BMJ 1997; 315: 540543.
21. Ferrandina G, Lauriola L, Zannoni GF et al. Expression of cyclooxygenase-2 (COX-2) in tumour and stroma compartments in cervical cancer: clinical implications. Br J Cancer 2002; 87: 11451152.[CrossRef][ISI][Medline]
22. Condon LT, Ashman JN, Ell SR et al. Overexpression of Bcl-2 in squamous cell carcinoma of the larynx: a marker of radioresistance. Int J Cancer 2002; 100: 472475.[CrossRef][ISI][Medline]
23. Holland JM, Arsanjani A, Liem BJ et al. Second malignancies in early stage laryngeal carcinoma patients treated with radiotherapy. J Laryngol Otol 2002; 116: 190193.[CrossRef][ISI][Medline]
24. Nix PA, Greenman J, Cawkwell L, Stafford N. Defining the criteria for radioresistant laryngeal cancer. Clin Otol (In Press).
25. Chan G, Boyle JO, Yang EK et al. Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck. Cancer Res 1999; 59: 991994.
26. Ranelletti FO, Almadori G, Rocca B et al. Prognostic significance of cyclooxygenase-2 in laryngeal squamous cell carcinoma. Int J Cancer 2001; 95: 343349.[CrossRef][ISI][Medline]
27. Gaffney DK, Holden J, Davis M et al. Elevated cyclooxygenase-2 expression correlates with diminished survival in carcinoma of the cervix treated with radiotherapy. Int J Radiat Oncol Biol Phys 2001; 49: 12131217.[ISI][Medline]
28. Kim YB, Kim GE, Cho NH et al. Overexpression of cyclooxygenase-2 is associated with a poor prognosis in patients with squamous cell carcinoma of the uterine cervix treated with radiation and concurrent chemotherapy. Cancer 2002; 95: 531539.[CrossRef][ISI][Medline]
29. Pruthi RS, Derksen E, Gaston K. Cyclooxygenase-2 as a potential target in the prevention and treatment of genitourinary tumors: a review. J Urol 2003; 169: 23522359.[ISI][Medline]
30. Liu XH, Yao S, Kirschenbaum A et al. NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells. Cancer Res 1998; 58: 42454249.
31. Petersen C, Petersen S, Milas L et al. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor. Clin Cancer Res 2000; 6: 25132520.
32. Silverstein FE, Faich G, Goldstein JL et al. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: a randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. JAMA 2000; 284: 12471255.
33. Kishi K, Petersen S, Petersen C et al. Preferential enhancement of tumor radioresponse by a cyclooxygenase-2 inhibitor. Cancer Res 2000; 60: 13261331.