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Abbreviations
TUR

transurethral resection

CIS

carcinoma in situ

CA

carbonic anhydrase

HIF

hypoxia- inducible factor

VHL

von Hippel- Lindau

VEGF

vascular endothelial growth factor.

INTRODUCTION

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES

It has been estimated that bladder cancer will affect 67 160 Americans in 2007 and that 17 120 will die of the disease [1]. The number of newly diagnosed cases has been increasing over the past decade, mainly due to more intensive evaluation of patients presenting with haematuria and irritative voiding symptoms, and improved physician and patient education [2]. The median age of newly diagnosed patients is 73 years, with men having a three-fold higher lifetime risk of developing bladder cancer than women [3]. About 90% of all bladder cancers are TCCs [4], and ≈70% of these tumours will be diagnosed at a noninvasive tumour stage (Ta, TIS, or T1). Despite aggressive treatment with a combination of transurethral resection (TUR) and intravesical agents such as BCG or mitomycin, recurrence rates are 50–70%. Furthermore, progression into muscle-invasive disease develops in 10–20% of patients who initially present with noninvasive tumours TCC [4]. Currently, there are few prognostic factors for the prediction of TCC recurrence or progression. Thus far, these factors are mainly clinical, and include stage, grade, tumour size, number of tumours, and associated carcinoma in situ (CIS). However, prediction of these endpoints remains difficult and integration of molecular markers such as p53 may be beneficial, but studies remain inconclusive [5].

Hypoxia is a common consequence of rapid growth of many vascular tumours and is an important regulator of gene expression. Carbonic anhydrase IX (CAIX) protein, an hypoxia-inducible member of the CA family that regulates intracellular pH during periods of hypoxia, is thought to play a role in the regulation of cell proliferation, cell adhesion, and tumour progression [6,7]. This brief review summarizes the current knowledge on CAIX expression in bladder TCC and its potential role as a marker of prognosis and as a therapeutic target.

GENERAL ROLE OF CAIX IN CANCER

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES

CAs are a large family of zinc metalloenzymes that are found in almost every organism. Today, there are 15 members of this family known, which differ with regards to tissue distribution and subcellular localization [7,8]. CAs catalyse the reversible reaction H2O + CO2 [LEFT RIGHT DOUBLE ARROW] H+ + HCO3-, which is crucial to a wide variety of processes including pH regulation.

CAIX belongs to the group of membrane-associated CAs. The term CA9 refers to the corresponding gene, which is located on chromosome 9p12–13 and consists of 1609 base pairs arranged in 11 exons. CA9 encodes for the 459 amino acid CAIX protein. CA9 is one of the 50 genes that are up-regulated by hypoxia-inducible factor 1α (HIF-1α). Therefore, a binding site for HIF-1α/hypoxia-responsible element is present in the CA9 promoter.

CAIX is not expressed in most benign organs and tissues. There is weak expression in the gastric mucosa, small intestine, biliary tract and seminal ducts [9]. However, CAIX is abundantly expressed as a direct consequence of hypoxia in numerous cancers [9]. In many of these cancers, the greatest staining intensities are on luminal surfaces or on surrounding areas of necrosis. Clear cell RCC is an exception to this general rule, and is the only cancer that shows a uniform staining pattern suggesting, probably that its expression in RCC is not a result of tumoral hypoxia but of a constitutively up-regulated HIF-pathway secondary to the inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene [6]. Studies show that high CAIX expression yields an aggressive tumour phenotype and poor prognosis in breast cancer [10], cervical cancer [11,12], non-small cell lung cancer [13], soft tissue sarcoma [14] and adenocarcinoma of the upper gastrointestinal tract [15,16], while the opposite has been noted in RCC [17,18].

EXPRESSION IN BLADDER CANCER

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES

Normal urothelial tissue [19,20] does not express CAIX. By contrast, expression is observed in 70–90% of TCCs [20,21], but rarely found in CIS. In 10 cases of CIS studied by Turner et al. [19], expression was weak in three and absent in seven of the specimens. In patients with metastatic TCC, simultaneously extirpated metastases show higher CAIX expression than the corresponding primary tumours [20].

Although expressed by most TCCs, expression frequency and intensity are usually low. Tumours stained by Hoskin et al. [22] showed an average stained tumour fraction of only 9%. Wykoff et al. [23] studied 14 TCCs derived from patients who had received pimonidazole before surgical excision. Although 12 of 14 specimens showed CAIX expression, the median percentage of tumour cells staining positive was only 5%. The authors compared expression levels with those of the bioreductive hypoxia marker pimonidazole. There was significant correlation between both markers, although expression was less strong for CAIX [23]. A similar relationship has been shown for CAIX and vascular endothelial growth factor (VEGF)-A mRNA levels [19].

Staining of CAIX is heterogeneous throughout each tumour [22]. Maximum staining is on the luminal surface of the papillary structures (Fig. 1). Additionally, CAIX expression is seen around areas of necrosis in invasive tumours or metastases (Fig. 2) [19,22,23].

image

Figure 1. Expression in superficial TCC on luminal papillary surfaces (×100).

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Figure 2. Lymph node metastasis of a high-grade TCC. Staining is only seen in regions bordering necrotic areas (×40).

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Studies suggest that CAIX is expressed in relation to stage and grade. Ord et al. [24] found that 13 out of 21 of the noninvasive, but only 3 out of 11 of the invasive tumours stained positive for CAIX. More recently, in a series of 98 patients with bladder cancer, the same group reported an increase in CAIX positivity among T1, T2, and T3 tumours, but expression levels decreased in T4 [25].

Our recent data shows that CAIX might be differentially regulated in low-grade and high-grade TCC [20]. In a series of 522 TCCs, 0% of normal urothelial samples, 85% of the grade 1 tumours, 79% of the grade 2 tumours, but only 63% of the grade 3 tumours stained positive for CAIX (P < 0.001). In contrast to our findings, one group did not show a significant relationship between CAIX expression and grade [25]. Overall, published data supports a role of CAIX as a diagnostic marker for TCC. CAIX might complement urinary cytology as a noninvasive marker to monitor for TCC because it is able to differentiate between normal urothelial cells and low-grade tumours. For example, in a small series of cytological samples, CAIX staining of urinary sediment could distinguish between benign papillary clusters and low-grade papillary tumours.

ROLE AS A PROGNOSTIC FACTOR

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES

Several studies on the role of CAIX as a prognostic factor in bladder cancer have been inconclusive. Most studies did not show a significant relationship, but all were limited by few samples and subsequent low statistical power. A large recent study from the author’s institution showed overwhelming prognostic significance in both superficial and muscle-invasive disease [20].

Ord et al. [25] reported on the role of hypoxia and necrosis in 98 patients with bladder cancer treated by cystectomy. The authors evaluated staining of CAIX, HIF-1α, HIF-2α, and Bcl2/adenovirus EIB 19 kDaA interacting protein 3 by immunohistochemistry. No association was found between CAIX and survival (P = 0.55). Moreover, none of these markers were retained as independent prognostic factors in multivariate analysis.

In a series of 49 cases reported by Turner et al. [19], recurrence- and progression-free survival in patients with low and high CAIX expression was similar. The authors correlated mRNA expression of VEGF-A and immunohistochemical expression of CAIX. Although there was a striking overlap, VEGF-A expression, but not CAIX, was predictive of time to recurrence and risk of stage progression.

Hussain et al. [26] investigated 57 patients with newly diagnosed TCC for CAIX expression and correlated their findings with survival. Tumours expressing CAIX weakly showed a trend towards shorter survival (P = 0.21). Stratified by stage, no significant association was found between CAIX and survival in superficial (P = 0.9) or invasive TCC (P = 0.94).

Hoskin et al. [22] studied CAIX expression in relation to survival in 64 patients treated by radiotherapy with carbogen and nicotinamide. Higher CAIX expression predicted worsened cancer-specific and overall survival. The 5-year overall survival rate for tumours expressing higher than the median CAIX value was 35% compared with 71% with low levels. Furthermore, CAIX expression was an independent prognostic factor in multivariate analysis (hazard ratio 3.21, 95% CI 1.16–10.22, P = 0.02), when CAIX and GLUT-1 expression were entered individually. However, the significance was lost when both variables were entered simultaneously.

Recently, we reported on survival of 351 patients undergoing surgical resection for bladder cancer [20]. For patients with Ta TCC undergoing TUR, higher CAIX expression (>45%) was associated with poorer recurrence-free survival (P = 0.02). In patients with T1 tumours, higher CAIX expression (>20%) also conveyed a worse prognosis with respect to recurrence-free (P < 0.001) and progression-free survival (P = 0.01). In patients undergoing cystectomy for muscle-invasive TCC, higher CAIX expression (>10%) was an independent prognostic factor of diminished overall survival (P = 0.002) [20]. Taken together, CAIX has been identified as an important predictor of the three survival endpoints in bladder cancer: recurrence, progression, and overall survival.

ROLE AS A POTENTIAL THERAPEUTIC TARGET

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES

Cornerstones in the treatment of bladder TCC include surgery, intravescial instillation, and systemic chemotherapy with or with no radiation. As CAIX is expressed in 70–90% of TCCs, but not in normal urothelial tissue, it represents a potential cancer-specific therapeutic target. First, particularly as CAIX is expressed on the luminal cell surface of the tumours, there may be a role as an intravesical-targeted agent for instillation therapy. For this purpose, baseline immunohistochemical staining of CAIX expression levels could be used to select patients and to determine whether the tumour can be targeted appropriately with a CAIX-directed approach. Second, there may be a role in systemic treatment for patients with metastatic disease. The chimeric monoclonal antibody G250, for example, has been extensively evaluated clinically in patients with RCC [27,28]. It was well-tolerated and showed promising antitumour effects [27]. A large randomized trial in an adjuvant setting is currently under way [29].

Recently, antibody-drug conjugates have gained attention for the treatment of advanced cancers [30]. In this concept, monoclonal antibodies are linked with cytotoxic agents, which specifically bind to target cells that express the antigen. Using this approach, the potential cytotoxic effect could be limited through targeted tumour delivery. For the treatment of bladder cancer, for example, G250 could be conjugated with a toxin or with a chemotherapeutic drug such as cisplatin.

Moreover, vaccine therapies have shown promise of efficacy in RCC [31–33]. Given that bladder cancer represents an immuno-sensitive disease responding to agents such as interferon α and BCG, administration of vaccines may also be an effective approach. Taken together, the role of CAIX as a therapeutic target in TCC is conceptually sound and warrants further investigation.

CONCLUSIONS

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES

CAIX is a bladder cancer-specific antigen that is not expressed in normal urothelial tissue but is expressed in 70–90% of TCCs. Expression is usually heterogeneous, with maximum staining seen on the luminal surfaces of the papillae and in perinecrotic areas. It appears that expression levels are related to stage and grade. Studies indicate that higher CAIX expression is associated with adverse prognostic features, such as increased recurrence and progression, and worse survival. CAIX may be exploited as a therapeutic target for both intravesical and systemic treatment. Thus, CAIX has important tripartite implications as a diagnostic, prognostic and therapeutic molecular marker of bladder cancer.

REFERENCES

  1. Top of page
  2. INTRODUCTION
  3. GENERAL ROLE OF CAIX IN CANCER
  4. EXPRESSION IN BLADDER CANCER
  5. ROLE AS A PROGNOSTIC FACTOR
  6. ROLE AS A POTENTIAL THERAPEUTIC TARGET
  7. CONCLUSIONS
  8. CONFLICT OF INTERESTS
  9. REFERENCES