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Hypoxia-regulated Carbonic Anhydrase-9 (CA9) Relates to Poor Vascularization and Resistance of Squamous Cell Head and Neck Cancer to Chemoradiotherapy

Tumour and Angiogenesis Research Group, Departments of Radiotherapy/Oncology, Pathology and Surgery, Democritus University of Thrace, Alexandroupolis 68100, Greece [M. I. K., A. G., E. S., K. S.]; Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic, 84246 [J. P.]; and Departments of Cellular Science and Institute of Molecular Medicine, Oxford Radcliffe Hospital, Headington, Oxford, OX3 9DS, United Kingdom [C. C. W., K. C. G., A. L. H.]

	ABSTRACT

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Purpose: Carbonic anhydrases are proteins involved in the catalytic hydration of carbon dioxide to carbonic acid. Recent studies show that carbonic anhydrase 9 (CA9) is up-regulated by hypoxia and that its immunohistochemical tissue distribution follows the distribution of the radiosensitizer pimonidazole (C. C. Wykoff et al., Cancer Res. 60: 7075–7083, 2001). Therefore, CA9 expression may show hypoxia levels of clinical importance.

Experimental Design: We assessed the expression of CA9 and the microvessel density (MVD; CD31-positive) in 75 locally advanced squamous cell head and neck cancers treated with concurrent chemoradiotherapy with carboplatin.

Results: Strong membrane/cytoplasmic CA9 expression, noted in 20/75 (26.6%) tumors, mainly occurred in tumors with very poor vascularization (expression in 63% versus 14%; P < 0.0001), was located around areas of focal necrosis, and was related to poor complete response rate (40% versus 70%; P = 0.02). These observations suggested that CA9 might be a marker of clinically important hypoxia. Combining the CA9 staining and the tumor angiogenicity (MVD), we identified three groups of patients: (a) hypoxic tumors; (b) euoxic highly angiogenic tumors; and (c) euoxic non-highly angiogenic tumors. Groups (a) and (b) had a very poor local relapse-free survival (P < 0.0001).

Conclusions: Stratification of patients undergoing radical radiotherapy using the CA9/MVD model may be useful for the individualization of therapeutic strategies combining antiangiogenesis and hypoxia targeting with radiotherapy.

	INTRODUCTION

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Hypoxia has been long considered a major cause of the failure of radiotherapy (1) . The development of simple and reliable tests to estimate the clinically relevant hypoxia is of importance for the identification of subgroups of patients that could benefit from hypoxia targeting therapeutic policies (2) . Assessment of tissue oxygen tension with polarographic electrodes or immunohistochemical detection of nitroimidazole accumulation is quite cumbersome to find a place in the clinical routine. Scintigraphic and magnetic resonance imaging of hypoxia are promising approaches, although as yet experimental. Nevertheless, all of these hypoxia detecting methods can only be applied in vivo and on a prospective basis.

In search of a simple test that would detect hypoxia even on archival tissue material, immunohistochemical detection of proteins induced by clinically relevant levels of hypoxia represents an appealing option. The transmembrane carbonic anhydrase group of proteins involved in the catalytic hydration of carbon dioxide to carbonic acid (3 , 4) is up-regulated by hypoxia, probably through the HIF-1² pathway (5) . In a previous study, Wykoff et al. (5) reported that CA9 localization showed a substantial, though incomplete, overlap with pimonidazole accumulation in patients with skin and bladder cancer. Therefore, the frame of hypoxia levels for CA9 induction may be of clinical relevance, at least in those tumors that do not express constitutively CA9 (i.e., renal clear cell carcinoma; Ref. 5 ).

In the present study, we assessed the expression of CA9 in archival tissue material from SCHNC. Our findings suggest that CA9 expression by cancer cells occurs mainly in very poorly vascularized tumors, is located around areas of necrosis, and strongly associates with resistance to radiotherapy.

	MATERIALS AND METHODS

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Archival paraffin-embedded biopsy material from 75 primary SCHNCs and 15 normal tongue, pharyngeal, and laryngeal mucosa were retrieved, and 2-µm tissue sections were cut on slides. All of the patients had locally advanced inoperable cancer (Tx/N2b-3 or T3–4/Nx stage) and were treated with conventionally fractionated radiotherapy (2 Gy/fraction, 5 fractions/week; total dose 68–72 Gy; LINAC 6 MV X-ray irradiation) concurrently with carboplatin (area under the curve, 5 every 4 weeks or area under the curve 1 from day 1 to day 9 and from day 31 to day 39 of radiotherapy). None of the patients underwent surgery. The follow-up of patients ranges from 1–108 months (median, 14 months). For patients alive the follow-up ranges from 6–108 months (median, 48 months). Thirty-one patients had pharyngeal cancer (oropharynx and hypopharynx), 32 had laryngeal cancer, and 12 patients had cancer of the maxillary antrum or well-differentiated squamous cell cancer of the nasopharynx.

Response to treatment was assessed with a computed tomography scan of the head-neck area 45–60 days after radiation treatment completion. Complete response was defined as the disappearance of all of the measurable lesions within 2 months after treatment completion, whereas partial response refers to a 50–95% reduction in tumor size. In the present study, minimal responders (tumor reduction between 25–50%) or patients with stable and progressive disease were considered in one group as "nonresponders."

Immunohistochemistry.
For the detection of CA9, we used the APAAP procedure and the mouse monoclonal antihuman CA9 antibody M75 (5) . Briefly, sections were dewaxed and rehydrated. After microwaving (4 min x 2) the primary antibody (dilution 1:50) was applied at room temperature for 90 min, and slides were washed in Tris-buffered saline. Rabbit antimouse antibody 1:50 (v/v) was applied for 30 min, followed by application of APAAP complex (1:1, v/v; Dako, Copenhagen, Denmark) for 30 min. After washing in Tris-buffered saline, the last two steps were repeated for 10 min each. The color was developed by 15-min incubation with new fuchsin solution. The specimens were scanned at low optical power (x40 and x100), and the percentage of cells with positive CA9 reactivity was assessed on all of the x200 fields (3–7 x 200 fields/case).

The JC70 monoclonal antibody (Dako) recognizing CD31 (platelet/endothelial cell adhesion molecule; PECAM-1) was used for microvessel staining using the APAAP method, as described previously (6) . The specimens were scanned at low optical power (x40 and x100), and microvessel counting was performed on x200 fields. Three areas (per case) of high vascularization were chosen for microvessel counting. The final MVD for a case was the mean value of the three appraised fields. Vessels with a clearly defined lumen or well-defined linear vessel shape but not single endothelial cells were taken into account for microvessel counting. Interobserver variability of MVD assessment was minimal as analyzed previously (6) . The MVD grouping of our cases was based on a previous study (6) . The 25^th, 50^th, and 75^th percentile of MVD was used to define four MVD categories: MVD1 (very low); MVD2 (low); MVD3 (medium); and MVD4 (high).

Statistical Analysis.
Statistical analysis and graphs were performed using the GraphPad Prism 2.01 and the Instat 3.0 packages (San Diego, CA).³ The Fisher’s exact t test or the Yates’ continuity corrected ² test was used for testing relationships between categorical tumor variables as appropriate. Nonparametric analysis was used to assess interobserver variability. Survival curves were plotted using the Kaplan-Meier method, and the log-rank test was used to determine statistical differences between life tables. The end points for analysis were the response rate, the local progression-free survival, and the OS starting from the last day of radiotherapy. Complete response rate was separately assessed from partial response rate, because head and neck cancer is a curable disease, and, thus, the partial response been always indicative of treatment failure. All of the Ps are two sided and Ps <0.05 were used for significance.

	RESULTS AND DISCUSSION

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CA9 Expression.
Strong cancer cell membrane and cytoplasmic CA9 expression was noted in 20/75 (26.6%) cases (Fig. 1) . These cases were considered as positive. In the remaining "negative" cases, CA9 reactivity was either absent or very weak cytoplasmic without membrane reactivity, confined in focal tumor areas. Positive staining, when present, was located around areas of focal necrosis. Because the specimens analyzed were bioptical, a direct analysis of CA9 expression with the extension of necrosis would not be reliable and was not attempted. A direct association of CA9 expression with necrosis in squamous cell lung cancer has been noted in a submitted study.⁴ Overall, the percentage of CA9-positive cells in tumor samples ranged from 0 to 60% (median, 0%; mean, 7.7%). In CA9-positive cases, the percentage of cells with CA9 reactivity ranged from 10 to 60% (median, 30%; mean, 29%). The percentage of cancer cells with positive cases (strong membrane and cytoplasmic staining) was recorded by two observers independently. Nonparametric analysis revealed overlapping results (P = 0.0001; r = 0.94), which is explained by the very clear membrane staining provided by the antibody. Normal head and neck tissues never expressed CA9, although a rather weak cytoplasmic expression of the normal mucosa cells located adjacent to tumors was noted occasionally (independently of the tumor CA9 expression), showing that normal tissues adjacent to tumors may suffer from hypoxia. Tumoral vessels and fibroblasts were occasionally positive.

View larger version (120K): [in this window] [in a new window]   Fig. 1. SSCHNC with strong membrane and cytoplasmic expression of CA9 located around areas of necrosis (x100).

Angiogenesis and CA9.
The expression of CA9 was mainly confined in tumors with very low MVD (MVD1 group; Fig. 2 ). The poor blood supply in these tumors probably accounts for a hypoxia-mediated induction of CA9. This finding also suggests that, in the majority of cases, quite a low number of vessels is adequate to provide a blood supply and oxygen tension enough to avoid the induction of CA9. The fact that: (a) some cases (7/19) with very low MVD did not express CA9 and that (b) some cases (8/56) with and intermediate or even high MVD expressed CA9 may show that, apart from the blood supply, the individual cancer cell metabolic demands for oxygen may also define the tumor oxygenation status. Moreover, intratumoral conditions that favor vascular collapse and reduced blood flow despite the high vascular density may also underlie the induction of CA9 in a subset of well-vascularized tumors.

View larger version (36K): [in this window] [in a new window]   Fig. 2. A, CA9 expression was significantly more frequent in tumors with very low MVD (<19 vessels/x200 optical field). B, percentage of cancer cells with strong CA9 reactivity was significantly higher in the group of tumors with very low MVD.

CA9 and Response to Chemoradiotherapy.
Tumors with CA9 expression had a significantly poorer complete response rate (40% versus 70.9%; P = 0.02; Table 1 ). This shows that the levels of hypoxia necessary for the induction of CA9 are clinically relevant to resistance to radiotherapy. Looking into the group of tumors with very low vascularization (MVD1; where CA9 was mainly expressed), all of the patients with negative CA9 expression responded completely to radiotherapy. This shows that CA9 expression is a more reliable marker of hypoxia as compared with low MVD. Even cases with a higher MVD (MVD2, 3, and 4) and CA9 expression had a poorer response rate, which shows that hypoxia levels in the range of CA9 induction, which may also occur despite the good vascularization, are related to resistance to radiotherapy.

Assuming that CA9 induction is a result of hypoxia in SCHNC, we used the CA9 expression and the angiogenic potential (as assessed with microvessel counting) to distinguish three different groups of tumors: (a) hypoxic tumors (any MVD with CA9 expression); (b) euoxic tumors with low/intermediate angiogenicity (CA9-negative, and MVD1, 2, and 3 cases); and (c) euoxic tumors with very high angiogenic potential (CA9-positive and MVD4 cases). Fig. 3 shows the LRFS and OS curves plotted for these three groups of patients, demonstrating that hypoxia assessed with the CA9 induction relates strongly to poor prognosis after definitive chemoradiotherapy. Moreover, highly angiogenic euoxic tumors similarly share a very poor outcome.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Kaplan-Meier LRFS (a) and OS (b) grouping cases according to their oxygenation status (as assessed by CA9; C, hypoxic versus A/B euoxic) and to their angiogenic potential (assessed by anti-CD31; A, euoxic/non-highly angiogenic versus B, euoxic/highly angiogenic).

Implications.
In conclusion, the present study suggests that CA9 is induced in poorly vascularized SCHNC and is located around areas of necrosis. Therefore, additional evidence that CA9 is induced by hypoxia in this tumor type is provided. Moreover, the hypoxia levels required for the induction of CA9 in SCHNC are clinically important, because CA9-expressing tumors were resistant to radiotherapy. Grouping SCHNC patients undergoing radiotherapy according to CA9 expression (used as a marker of hypoxia) and to MVD (used as a marker of tumor angiogenicity), seems to have a strong predictive value. Furthermore, individualized therapeutic strategies may be guided by such a stratification. Combination of radiotherapy with bioreductive drugs, hyperthermia, or even heavy particle irradiation may improve the bad results of radiotherapy in hypoxic (CA9+) tumors. On the other hand, antiangiogenesis policies combined with radiotherapy may be of importance for the effective eradication of highly angiogenic tumors. Euoxic tumors (assessed as CA9-negative cases) sharing a low or intermediate angiogenic potential seem to be highly sensitive to standard chemoradiotherapy schedules. Application of the model in tissue material from patients recruited in large randomized studies that ended in frustration (17 , 18) may reveal the subgroups of patients that can benefit from combinations of radiotherapy with various chemical or physical radiosensitizers.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Tumour and Angiogenesis Research Group, 18 Dimokratias Avenue, Iraklion 71306, Crete, Greece. Phone: 0030-932-480808; Fax: 0030-81-284661; E-mail: targ{at}her.forthnet.gr

² The abbreviations used are: HIF, hypoxia-inducible factor; CA9, carbonic anhydrase 9; SCHNC; squamous cell head and neck carcinoma; APAAP, alkaline phosphatase/antialkaline phosphatase; MVD, microvessel density; LRFS, local relapse-free survival; OS, overall survival.

³ Internet address: www.graphpad.com.

⁴ A. Giatromanolaki, M. I. Koukourakis, E. Sivridis, J. Pastorek, C. C. Wykoff, K. C. Gatter, A. L. Harris. Expression of hypoxia inducible carbonic anhydrase (CA9) relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer. Cancer Res., in press, 2001.

Received 4/27/01; revised 6/28/01; accepted 7/31/01.

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