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7 Role of cyclooxygenase2 expression in colorectal cancer
9
T'
Role of Cyciooxygenase2 Expression in Colorectai Cancer Sven Petersen
Introduction Since their discovery four decades ago prostaglandins (PGs) have been assumed to be precursors for formation and growth of malignant tumors. Thus, the manipulation of PG pathways and their impact on cancer growth and treatment have become important in cancer research. The purpose of this study is to determine whether the expression pattern of cyclooxygenase2 (COX2) in a series of human rectal tumors is linked to the outcome in a well-defined cohort of patients who underwent surgery for rectal cancer.
Prostaglandins
including blood vessel tone, platelet aggregation, and immune responses. The maintenance of the gastric mucosa is regulated by PGs as well as regulation of cell growth and differentiation. PGs are involved in many pathologic conditions such as inflammatory reactions or rheumatoid disease (Kargman et al., 1995; Kutchera et al., 1996). PGs have also been implicated in cancer development with a number of tumor types found to produce more PGs than the normal tissues from which they arise (Hida et al., 1998; Kutchera et al., 1996). PGs stimulate angiogenesis and, in addition, are vasoactive agents, which may influence tumor growth and response to cytotoxic agents (Ziche et al., 1982). Therefore, tumor characteristics such as rapid growth and metastatic spread might be linked to the effect of increased intratumoral PG levels.
PGs became of interest for experimental research after they were discovered as a group of mediators in the 1960s. The precursor of all PGs is arachidonic acid, a polyunsaturated fatty acid, which is liberated from the cell membrane phospholipids by the phosp h o l i p a s e A 2. Arachidonic acid then is catalyzed by the COX enzyme to an unstable precursor PG G 2. This intermediate form is transferred to PG H 2, which is the precursor for all resulting prostaglandins including PG E 2, PG Fza, PG D 2, PG I 2, thromboxane TX A 2 and TX B 2. Prostaglandins serve as critical mediators in mammalian physiology affecting a variety of functions,
COX1 and COX2 The rate-limiting enzyme in the synthesis of PGs from arachidonic acid is COX. The PG synthase COX1 was first cloned in 1988 (Fosslien, 2000). Since 1991 it has been known that two isoforms of COX exist (Kujubu et al., 1991). COX1 is constitutively expressed in most tissues and mediates the synthesis of PGs required for normal physiologic functions. In contrast, COX2 is typically not expressed or expressed at relatively low
Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 2: Molecular Pathology, Colorectal Carcinoma, and Prostate Carcinoma
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levels in undisturbed healthy tissues but is inducible by an assortment of agents including proinflammatory stimuli, mitogens, and/or hormones depending on the tissue (DuBois et al., 1994; Kujubu, et al., 1991). COX1 and COX2 are encoded by genes mapping to chromosomes 9 and 1, respectively. The COX1 gene extends for 22 kilobases (kb) and includes 11 exons, producing a 2.7 kb message. COX2 is an 8.3 kb gene of 10 exons that generates a 4.3-4.5 kb message. Although the intron/exon structure of the genes is nearly identical and the encoded proteins are ~70% homologous, the regulatory elements within the genes are quite different. Whereas COX1 represents a housekeeping gene, which lacks a TATA box (Kraemer et al., 1992), the promotor of the immediate-early gene COX2 contains a TATA box and binding sites for several transcription factors including nuclear factor-~B, the nuclear factor for interleukin-6 expression and the cyclic adenosine monophosphate response element binding protein (Appleby et al., 1994). COX2 was shown to be moderately overexpressed in a large variety of tumor types including head and neck, colon, breast, and pancreatic cancers. In addition, there is considerable evidence available indicating that COX2 promotes carcinogenesis and growth of established tumors (Eberhart et al., 1994; Fujita et al., 2000; Taketo, 1998; Tsujii et al., 1997). In most tumors, COX2 expression was detected in --~40-80% of tumor cells. However, even some normal tumor cell infiltrates, particularly epithelial cells, may express COX2. Although the relationship between COX2 and tumor aggressiveness is not fully established, it seems that COX2 overexpression is related to poor patient prognosis and enhanced propensity for metastatic spread (Achiwa et al., 1999; Sheehan et al., 1999). Enhanced COX2 expression might also be related to the grade of differentiation of the tumors. For instance, in some well-differentiated adenocarcinomas of the lung an increased COX2 level was found (Wolff et al., 1988), but COX2 was also found in other forms of lung cancer (Hida et al., 1998; Khuri et al., 2001). Probably the largest number of publications deals with COX2 expression in colorectal adenomas and carcinomas, and in this case the potential clinical application of nonsteroidal anti-inflammatory drugs has been debated for decades. Most of colorectal cancer cells express COX2. The level of COX2 expression varies depending on the detection methods, and the published data suggest that according to the tumor site, COX2 expression might be more pronounced in rectal tumors compared to the colonic tumors (Dimberg et al., 1999; Sano et al., 1995). It is controversial whether COX2 expression in itself is a prognostic factor for local recurrence and/or
II Colorectal Carcinoma
survival of patients with colorectal cancer. Some authors found a significantly higher incidence of local recurrence and increased cancer-specific mortality associated with higher COX2 expression in tumor cells (Tomozawa et al., 2000), whereas others could not confirm these observations (Fujita et al., 1998). The aim of this synopsis is to determine the influence of COX2 expression in colorectal carcinoma on tumor recurrence and survival. In this context the COX2 expression in tumors of patients treated surgically for rectal cancer in our department were analyzed. The COX2 immunohistochemistry staining technique will be described in detail. Of special interest is to analyze whether there were significant differences in the group with low COX2 expression compared to the high COX2 group with reference to well-known prognostic factors (Fielding et al., 1991). In addition, our data will be compared with currently available data on COX2 expression in colorectal cancer.
COX2 Immunohistochemistry MATERIALS
1. Tissue samples embedded in paraffin. 2. For deparaffinization: 2X xylene 400 ml; isopropanol, 2X 400 ml; ethanol solution 96% in distilled water, 2X 400 ml; ethanol solution 70%, 2X 400 ml; ethanol solution 50%, 2X 400 ml; distilled water, 2X 400 ml. Phosphate buffer saline (PBS): 100 mg anhydrous calcium chloride, 200 mg monobasic potassium phosphate, 100 mg magnesium chloride, 8 g sodium chloride, and 2.16 g dibasic sodium phosphate; bring volume to 1 L with deionized distilled water (pH 7.4). 3. Solution for unmasking the antigens: citrate buffer solution: 450 ml distilled water, 9 ml of 100 mmol/L citrate, and 41 ml of 100 mmol/L sodium citrate. Tris buffer solution: 900 ml of 154 mmol/L NaC1 and 100 ml Tris-buffer stock solution. 4. For endogenous peroxidase blocking: --~400 ml of 3% hydrogen peroxide solution. 5. Incubation with primary antibody. Tris buffer solution: 900 ml of 154 mmol/L NaC1 and 100 ml Tris-buffer stock solution. Primary antibody" rabbit polyclonal antibody specific for human recombinant prostaglandin H synthase Form-2 (COX2) diluted 300-fold in antibody dilution (Dako, Code No. $3022, DakoCytomation GmbH, Hamburg, Germany). The antibody was provided by Oxford Biomedical Research Inc., Michigan. 6. For immunostaining, the Dako kit EnVision System was used. The immunostaining kit was provided by DakoCytomation GmbH. Incubation with secondary antibody alkaline phosphatase labeled polymer and
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7 Role of COX2 Expression in Colorectal Cancer substrate chromogen solution (Fast Red) was used. Distilled water, 2X 400 ml. 8. Counterstaining: hematoxylin. 9. For the dehydration of the samples, the same materials as in Step 2 were used.
METHODS Tissue samples were fixed with 4% formaldehyde in PBS, embedded in paraffin, and cut in 4 Bm thick sections. The sections were deparaffinized, hydrated through xylene and ethanol, and microwaved. The following steps were performed: 1. Rinse the samples two times in xylene for 10 min each. 2. Rinse the samples two times in isopropanol for 2-3 min each. 3. Hydrate the samples by rinsing first two times in 96% ethanol for 2-3 min each, followed by rinsing in 70% ethanol once for 2-3 min and 50% ethanol once for 2-3 min. Finally, rinse the samples two times in distilled water for 2-3 min each. 4. Microwave the samples two times for 5 min each. 5. Rinse the samples in Tris buffer two times for 5 min each. 6. Endogenous peroxidase was blocked by immersion of the samples in 3% hydrogen peroxide for 30 min at room temperature. Estimated 100 B1 peroxidase solution for each sample. 7. Rinse the samples in Tris buffer two times for 5 min each. 8. Incubation with the COX2 antibody, for this rabbit polyclonal antibody specific for human COX2 (Oxford) diluted 300-fold, was applied and sections were incubated at 4~ overnight. Estimated 100 ~tl antibody solution for each sample. 9. Rinse the samples in Tris buffer two times for 5 min each. 10. Incubation with alkaline phosphatase labeled polymer from the Dako EnVision kit for 30 min at room temperature. Estimated 100 B1 polymer solution for each sample. 11. Rinse the samples in Tris buffer two times for 5 min each. 12. Apply substrate chromogen solution (Fast Red) from the Dako EnVision kit for 8 min at room temperature. Estimated 100 B1 chromogen solution for each sample. 13. Rinse the samples in distilled water two times for 5 min each. 14. The sections were counterstained with hematoxylin (HE); incubate the samples shortly in the HE solution just until the nuclears start staining (--~5-10 seconds).
15. Rinse the samples with room temperature disfilled water until the HE staining changes color from brown to blue. 16. Dehydrate the samples by rinsing two times in distilled water for 2-3 min each. Apply 50% ethanol for 2-3 rain once and ethanol 70% for 2-3 rain one time. Rinse the samples twice for 2-3 min in 96% ethanol solution. After that, rinse the samples with isopropanol twice for 2-3 min, and finally rinse the samples twice in xylene for 10 min each. 17. Mount the samples. Nonimmunized rabbit serum was used as negative control.
Evaluation of COX2 Immunostaining The specimens immunostained for COX2 were evaluated according to the intensity and extent of positive reaction of tumor cells on a semiquantitative scale. The number of stained versus not stained epithelial cells was counted in at least 500 cancer cells in the area of the most intense staining. A labeling index of COX2 staining was calculated by dividing the number of stained cells by all counted tumor cells. The median labeling index was the base of the calculations for the Chi-square test and the univariate and multivariate analyses; patients were classified in two groups, one above and one below the median COX2 labeling index. In addition, in all tumor samples the staining intensity of COX2 was divided semiquantitatively into grades 1-4, from grades 1 (low), 2 (mild), 3 (moderate), to 4 (high). In 46 of all specimens simultaneous evaluation of the staining intensity of normal tissue mucosa was possible.
Patient Samples Specimens from 62 consecutive patients with International Union Against Cancer (UICC) stage I-III rectal cancer received radical surgical treatment at the Department of General and Abdominal Surgery at the Hospital Dresden-Friedrichstadt, in the period 19951996, were evaluated. None of the patients underwent neoadjuvant chemotherapy or radiotherapy. The mean follow-up period was 42 months (standard deviation [SD] +17 months). The tumor was curatively resected in all patients; however, no mesorectal excision was performed in general. The mean number of examined lymphnodes was 10 (SD +8).
Survival The survival of patients was quantified in two different endpoints: overall survival and disease-specific survival. The overall survival excluded all dead
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patients independent of cause of death. The diseasespecific survival was defined as cumulative survival for patients with curative resection, excluding patients who died from cancer-related causes, with a proven local recurrence, distant metastasis, or both. Survival time was counted starting at the day of surgery.
Local Recurrence and Distant Metastasis All 62 patients had a curative resection, defined as complete removal of all macroscopically evident tumor and cancer-free resection margins on histologic examination. Patients who developed a histologically proven local recurrence, an anastomotic recurrence, or combined local recurrence with distant metastasis were included in the group of local recurrence. Distant metastasis was defined as newly discovered evidence of tumor recurrence using standard follow-up for colorectal cancer. Pulmonary metastases were detected by routine chest X-ray.
Tumor Classification and Histologic Categorization According to the World Health Organization (WHO) categorization, only adenocarcinomas (International Classification of Diseases Manual [ICDM] 8140/3 or 8480/3) were included in this study. Mucinous tumors (ICDM 8480/3) were defined as more than 50% mucin in the extracellular matrix. The tumor classification was based on the TNM (tumor, lymph nodes, metastasis) staging system, including L category for lymph vessel invasion and the V1 classification for microscopic venous invasion (Hermanek et al., 1997).
Statistical Methods Statistical analysis was performed using the SPSS 10.0.7 software package (SPSS, Inc., Chicago, IL). Actuarial survival curves were calculated and plotted according to the Kaplan-Meier life-table method. For univariate analysis, comparison between the survival curves was made using the log-rank test. Variables with p-value less than 0.05 were considered to be significant.
RESULTS AND DISCUSSION
Tumor Staining All of the 62 specimens were stained positively for COX2 with a cytoplasmic immunoreactivity. There was a typical staining pattern of granular immunoreactivity in the apical part of the epithelial
Figure 30 COX2 staining in a highly differentiated tubulopapillar rectal adenocarcinoma (G1): intensive immunoreactivity in perinuclear granular of the epithelial tumor cells, nuclei with hematoxylin counterstaining (original magnification 100X). cells (Figure 30). No staining of the nucleus was observed. This is in agreement with other publications, which also found the staining pattern predominantly localized in the cytoplasm and the nuclear envelope (Tomozawa et al., 2000; Yamauchi et al., 2002). In this study, no staining of stroma cells was observed. This finding is supported by other authors who also found no stroma staining (Yamauchi et al., 2002). In contrast, there are also publications showing COX2 staining in stroma cells (Konno et al., 2002; Tomozawa et al., 2000). The median COX2 labeling index was 0.58 (SD __.0.25). Low staining intensity was observed in 10 specimens (16%); it was mild in 18 (29%), moderate in 28 (45%), and high in 6 cases (10%).
Mucosa Staining In 46 specimens (74%), mucosa could be evaluated. In the normal tissue the COX2 staining was evaluated as high in 1 specimen (2%); the staining was moderate in 19 cases (41%), mild in 18 cases (39%), and low in 8 specimens (17 %). The distribution of staining in tumor versus normal mucosa showed no difference. There was an increase of staining intensity from the distant parts of the mucosa to the tumor-adjacent mucosal areas in 17 cases (37%). These findings are in good agreement with other studies, which found COX2 staining of adjacent mucosa only in a minority of samples
7 Role of COX2 Expression in Colorectal Cancer
(Tomozawa et al., 2000; Zhang and Sun, 2002). However, it is emphasized that COX2 is eventually detectable in normal tissue. Thus, the simple paradigm that COX2 is found only in inflammatory- and cancer-related tissues is not always true. Whether this investigation is of relevance needs to be evaluated in further studies.
COX2 as Prognostic Factor for Survival The follow-up of 62 patients with rectal carcinoma revealed that 25 patients (40.3%) died. Using the median labeling index to distinguish low from high COX2 expression for survival analysis showed that 12 patients died after a mean of 48.6 months in the group of low COX2 expression and 13 patients died in the overexpression group after 44.2 months. These results are obviously not significant in the KaplanMeier survival statistics (p = 0.69). However, using a much higher threshold of a labeling index of 0.74 for COX2 overexpression provided significant results in the survival analysis, where 13 patients in the overexpression group only had a follow-up of 34.6 months compared to 49.7 months of survival in 49 patients with low COX2 expression ( p < 0.01). In the case of colorectal cancer and survival, a large variety of potential prognostic factors are available. Besides the well-established prognostic factors, the number of biologic and molecular markers that characterize carcinomas is increasing (Ratto et al., 1998). In addition, there are surgery-related factors, such as the extent of mesorectal excision in rectal surgery, the surgeon himself, or complicating events, which affect prognosis (Hermanek, 1999a; Marusch et al., 2002).
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Thus, Hermanek postulated the following recommendation for prognostic factors using immunohistochemistry: 1) standardization of methods to increase the acceptance of potential prognostic or predictive factors; 2) analysis of potential new markers must include the established prognostic factors with the highest statistical power (pT, pN, UICC stage, and curative resection R) (comments in Petersen et al., 2001). According to the value of prognostic factors, the Colorectal Working Group published the "American Joint Committee on Cancer Prognostic Factors Consensus Conference." In this survey, prognostic factors were classified into four groups (Compton et al., 2000). Category I includes prognostic factors that are well evaluated by published data and on which treatment strategies are based and which can modify the established TNM classification. In category IIa, factors from clinical or histologic evaluations with prognostic value might be added to the histopathologic characterization tumors. Category IIb includes well-studied prognostic factors that are not sufficiently established for category I or IIa. Prognostic variables, which are not yet established to meet criteria for category I and II, are summarized in category III. Category IV includes prognostic factors that show no consistent prognostic significance. The most important prognostic factors in category I are variables, which are included into TNM classification and the UICC stage (Compton et al., 2000; Fielding et al., 1991; Hermanek, 1999b). According to relevance of COX2 expression and survival analyses only data on immunohistochemistry (IHC) evaluation of COX2 are available (Table 4). Three studies found significant impact of COX2
Table 4 Published Data of Prognostic Influence of COX2 in Colorectal Cancer COX2 Antibody Detection (Dilution)
Cutoff (No. Patient: Negative versus Positive)
Overall Survival
DiseaseSpecific Survival
Author
Patients
Therapy
Konno et al. (2002) Masunaga et al. (2000)
56 100
IHC IHC
IBL (1:20) Cayman (1:300)
>5% (42 versus 14) >0% (24 versus 76)
Significant Significant
n.s. n.s.
13hd et al. (2003)
61
Surgery Surgery (n = 58 postoperative chemotherapy) Surgery
IHC
Cayman (1:500)
Insignificant
n.s.
Sheehan et al. (1999) Tomozawa et al.
76
Surgery
IHC
Cayman (1:500)
Low/moderate versus high/very high (n.s.) > 1% (14 versus 62)
Significant
n.s.
63
Surgery
IHC
IBL (1:40)
>50% (50 versus 13)
n.s.
Significant
232
Surgery
IHC
Alexis (1:1000)
>70% (66 versus 166) n.s.
Significant
112
n.s.
IHC
Dako (1:400)
n.s. (31 versus 81)
n.s.
(2000) Yamauchi et al. (2002) Zhang et al. (2002)
IHC, immunohistochemistry;n.s., not stated.
Insignificant
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expression on the overall survival (Konno et al., 2002; Masunaga et al., 2000; Sheehan et al., 1999); two additional studies provided evidence that COX2 overexpression reduced the disease-specific survival (Tomozawa et al., 2000; Yamauchi et al., 2002). Their results are inconsistent with the results reported here. Using the median COX2 labeling index as a cutoff point, no significant influence on local recurrence or survival was found. The data presented here, however, are in good agreement with the results presented by Zhang et al. (2002) and (Jhd et al. (2003). In a study including 112 patients with colorectal cancer, Zhang found no impact of COX2 expression on survival (Zhang and Sun, 2002). The authors concluded that COX2 overexpression is more likely linked to cancer differentiation rather than to prognostic value. Ohd et al. also could not confirm the relevance of COX2 overexpression on overall survival in 61 patients with colorectal cancer; they only found an impact of COX2 on survival in a subgroup of UICC-stage II patients (Ohd et al., 2003). The results with IHC can vary because of different antibodies, variations in the technique of incubation and antigen fixation, subjectivity in scoring, and absence of uniform cutoff value for definition of positive tumors. Accordingly, reasons for divergent results from different publication are mainly the result of differences of the immunohistochemical staining techniques (Garewal et al., 2003). As stated in Table 4, techniques for assessing the COX2 activation vary extensively (e.g., dilution of the antibody or the incubating time). In this study the Oxford polyclonal antibody was used. In contrast, other published data on COX2 in colorectal cancer and prognostic evaluation used other antibodies. Two studies used the IBL antibody (Konno et al., 2002; Tomozawa et al., 2000); three used the Cayman antibody (Masunaga et al., 2000; Ohd et al., 2003; Sheehan et al., 1999). In one study the Dako antibody was used (Zhang and Sun, 2002); in another study the staining analysis was performed using the Alexis antibody (Yamauchi et al., 2002). Another methodologic problem is the cutoff point to distinguish COX2 overexpression. The threshold varies substantially among different studies. Masunaga et al. (2000) and Sheehan et al. (1999) considered tumors with a minimal COX2 staining as positive. In contrast, Tomozawa et al. (2000) regarded IHC as COX2 overexpressed when 50% of all tumor cells showed staining pattern. Using the median labeling index as cutoff, the data presented here provide no impact of COX2 expression on local recurrence or overall survival. In contrast, there was a significant increased number of isolated pulmonary metastasis in the COX2 overexpression group.
II C o l o r e c t a l C a r c i n o m a
Changing the cutoff point of the labeling index to 0.74, only 13 patients showed COX2 overexpression. However, in this group the disease-specific and the overall survival were significantly decreased. This result of negative impact of very high COX2 expression is in good agreement with other studies. Konno et al. (2002) and Masunaga et al. (2000) found also a decreased overall survival associated with COX2 overexpression. Tomozawa et al. (2000) and Yamauchi et al. (2002) were able to show this effect of high COX2 levels on disease-specific survival. Thus, low COX2 expression in colorectal tumors does not seem to be linked to malign potential of carcinoma. In contrast, severe COX2 overexpression indicates increased malign potency of a tumor and an increased risk of potential metastatic spread. Only a small number of data is published according to the relevance of COX2 overexpression on local recurrence of colorectal cancer (Tomozawa et al., 2000). The data presented here provide evidence that COX2 overexpression is not linked to local failure following curative resection of rectal carcinoma.
COX2 and Metastasis One of the potential reasons of decreased survival associated with COX2 overexpression is an increased capability of the overexpressing tumor for metastatic spread. The data presented here show a higher incidence of lung metastasis in the group with increased levels of COX2 expression in the tumor samples. Using the median labeling index of 0.58, isolated lung metastasis was observed in five patients; all of these metastatic events happened in the overexpression group, which was obviously statistically significant in the Kaplan-Meier analysis (p = 0.04). In eight patients the rectal carcinoma recurred locally~five times in the lower expression group and only three times in the group with COX2 overexpression. These results were insignificant ( p = 0.41). The outcome presented here gives evidence that increased levels of COX2 expression in rectal carcinoma are associated with higher potency of hematogenous metastatic spread. The latter effect of COX2 is most likely linked to higher levels of PGs that are known to cause increased metastatic events (Honn et al., 1981). Metastatic events are the result of a complicated interaction between tumor and host. Primary tumor growth is followed by tumor cell translation to the location of metastasis; this event is closely related to angiogenesis (Hejna et al., 1999; Liotta et al., 1993). Thus, increased COX2-associated neoangionenesis might explain higher rates of hemateneous metastasis
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7 Role of COX2 Expression in Colorectal Cancer in tumors with COX2 overexpression (Cianchi et al., 2001; Costa et al., 2002; Gallo et al., 2001). Cianchi et al. (2001) reported a strong correlation between COX2 and vascular endothelial growth factor (VEGF) expression in human colorectal cancer specimens, and Gallo et al. (2001) showed head and neck tumors with high levels of VEGF and COX2 expression and significantly higher vascularization. Tomozawa et al. (2000) also reported increased rates of hematogenous metastasis in patients with COX2 overexpression in the tumor samples. These data were supported by experimental results from the same group. In a mouse model, they evaluated the number of lung metastases following intravenous tumor injection. The number of pulmonary metastases was significantly reduced by application of selective COX2 inhibitor (Tomozawa et al., 1999). The biologic background of the influence of COX2 expression on tumor growth and metastatic potential is a focus of investigation. There is evidence that high COX2 expression is associated with mutant p53 (Leung et al., 2001) or with EGF and nuclear factor-rJ3 expression (Saha et al., 1999; Sato et al., 1997). In addition, data are available from cell culture and in vivo studies indicating that elevated levels of COX2 promote angiogenesis, which might influence the metastatic behavior of tumor cells (Kishi et al., 2000; Sawaoka et al., 1999). Another mechanism that might be involved in COX2-associated metastatic spread is the connection of COX2 and the matrix-metalloproteinase 2 (MMP-2). MMP-2 modulates cell surface integrity and was shown to be associated with increased metastatic spread in a variety of tumors (Baker et al., 2000; Barozzi et al., 2002). Using COX2 transfected colorectal cancer cell in in vitro experiments it was shown that COX2 induced increased MMP-2 activity (Tsujii et al., 1997). Other metastasis-inducing factors that might be linked to COX2 overexpression are urokinase-type plasminogen activator (uPA) and interleukins (Fosslien, 2000; Konno et al., 2002). Because there is increasing emphasis to stratify treatment modalities according to molecular parameters, the critical evaluation of prognostic molecular factors has become essential (Petersen et al., 2001). According to COX2 overexpression in tumor samples, and the data presented here, the conclusion can be drawn that COX2 overexpression is linked to increased tumor aggressiveness. However, the immunohistochemical detection of COX2 is not yet an established prognostic factor. According to the American Joint Committee on Cancer Prognostic Factors Consensus Conference, at present COX2 overexpression can be classified as level IIb to III prognostic factor (Compton et al., 2000).
CONCLUSIONS The data presented here indicate that COX2 overexpression, detected with IHC analysis, has a marginal impact on the survival of surgically treated patients with colorectal cancer, but there is need for standardization of the technical approach to assess COX2 overexpression. Presently, there is not enough evidence that COX2 mutation is a predictive factor for response to adjuvant treatment. One of the major questions is how to treat patients with an immunohistochemical COX2 overexpression, cannot be answered sufficiently from the data. The role of COX2 as a predictive factor in colorectal cancer, therefore, needs to be addressed in appropriate clinical trials. Since selective COX2 inhibitors became available, the detection of COX2 expression has played an emerging role in the stratification of treatment with COX2 inhibitors in human malignant tumors. Experimental data show that COX2 inhibition, especially in combination with other treatment modalities, might be one part of cancer treatment in the future (Milas, 2001; Petersen et al., 2000). Studies are ongoing to evaluate the role of COX2 inhibition in human malignant neoplasms (Chang, 2002; VICTOR, 2000). The results of these studies will soon give evidence whether COX2 needs to be focused more in cancer treatment.
Acknowledgments I gratefully acknowledge the excellent technical assistance of Gunter Haroske and Mrs. Blumentritt from the Department of Pathology; Georg Schmorl, General Hospital DresdenFriedrichstadt; and Cordula Petersen and Michael Baumann from the Department of Radiotherapy and Oncology, University Dresden, Germany, in the immunohistochemistry studies.
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190 Cianchi, E, Cortesini, C., Bechi, E, Fantappie, O., Messerini, L., Vannacci, A., Sardi, I., Baroni, G., Boddi, V., Mazzanti, R., and Masini, E. 2001. Up-regulation of cyclooxygenase 2 gene expression correlates with tumor angiogenesis in human colorectal cancer. Gastroenterology 121:1339-1347. Compton, C., Fenoglio-Preiser, C.M., Pettigrew, N., and Fielding, L.E 2000. American Joint Committee on Cancer Prognostic Factors Consensus Conference: Colorectal Working Group. Cancer 88:1739-1757. Costa, C., Soares, R., Reis-Filho, J.S., Leitao, D., Amendoeira, I., and Schmitt, EC. 2002. Cyclo-oxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer. J. Clin. Pathol. 55:429-434. Dimberg, J., Samuelsson, A., Hugander, A., and Soderkvist, E 1999. Differential expression of cyclooxygenase 2 in human colorectal cancer. Gut 45:730-732. DuBois, R.N., Awad, J., Morrow, J., Roberts, L.J., 2nd, and Bishop, ER. 1994. Regulation of eicosanoid production and mitogenesis in rat intestinal epithelial cells by transforming growth factor-alpha and phorbol ester. J. Clin. Invest. 93:493-498. Eberhart, C.E., Coffey, R.J., Radhika, A., Giardiello, EM., Ferrenbach, S., and DuBois, R.N. 1994. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 107:1183-1188. Fielding, L.E, Arsenault, EA., Chapuis, EH., Dent, O., Gathright, B., Hardcastle, J.D., Hermanek, E, Jass, J.R., and Newland, R.C. 1991. Clinicopathological staging for colorectal cancer: an International Documentation System (IDS) and an International Comprehensive Anatomical Terminology (ICAT). J. Gastroenterol. Hepatol. 6:325-344. Fosslien, E. 2000. Molecular pathology of cyclooxygenase-2 in neoplasia. Ann. Clin. Lab. Sci. 30:3-21. Fujita, M., Fukui, H., Kusaka, T., Ueda, Y., and Fujimori, T. 2000. Immunohistochemical expression of cyclooxygenase (COX)-2 in colorectal adenomas. J. Gastroenterol. 35:488-490. Fujita, T., Matsui, M., Takaku, K., Uetake, H., Ichikawa, W., Taketo, M.M., and Sugihara, K. 1988. Size- and invasiondependent increase in cyclooxygenase 2 levels in human colorectal carcinomas. Cancer Res. 58:4823-4826. Gallo, O., Franchi, A., Magnelli, L., Sardi, I., Vannacci, A., Boddi, V., Chiarugi, V., and Masini, E. 2001. Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis. Neoplasia 3:53-61. Garewal, H., Ramsey, L., Fass, R., Hart, N.K., Payne, C.M., Bernstein, H., and Bernstein, C. 2003. Perils of immunohistochemistry: Variability in staining specificity of commercially available COX-2 antibodies on human colon tissue. Dig. Dis. Sci. 48:197-202.
Hejna, M., Raderer, M., and Zielinski, C.C. 1999. Inhibition of metastases by anticoagulants. J. Natl. Cancer Inst. 91:22-36. Hermanek, E 1999a. Impact of surgeon's technique on outcome after treatment of rectal carcinoma. Dis. Colon Rectum 42:559-562. Hermanek, E 1999b. Prognostic factor research in oncology. J. Clin. Epidemiol. 52:371-374. Hermanek, P., Hutter, R.V.E, Sobin, L.H., Wagner, G., and Wittekind, C. 1997. TNM atlas, 4th Edition. Berlin, Heidelberg, New York: Springer. Hida, T., Yatabe, Y., Achiwa, H., Muramatsu, H., Kozaki, K., Nakamura, S., Ogawa, M., Mitsudomi, T., Sugiura, T., and Takahashi, T. 1998. Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res. 58:3761-3764.
II Colorectal C a r c i n o m a Honn, K.V., Bockman, R.S., and Marnett, L.J. 1981. Prostaglandins and cancer: a review of tumor initiation through tumor metastasis. Prostaglandins 21:833-864. Kargman, S.L., O'Neill, G.P., Vickers, P.J., Evans, J.E, Mancini, J.A., and Jothy, S. 1995. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res. 55:2556-2559. Khuri, ER., Wu, H., Lee, J.J., Kemp, B.L., Lotan, R., Lippman, S.M., Feng, L., Hong, W.K., and Xu, X.C. 2001. Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I nonsmall cell lung cancer. Clin. Cancer Res. 7:861-867. Kishi, K., Petersen, S., Petersen, C., Hunter, N., Mason, K., Masferrer, J.L., Tofilon, EJ., and Milas, L. 2000. Preferential enhancement of tumor radioresponse by a cyclooxygenase-2 inhibitor. Cancer Res. 60:1326-1331. Konno, H., Baba, M., Shoji, T., Ohta, M., Suzuki, S., and Nakamura, S. 2002. Cyclooxygenase-2 expression correlates with uPAR levels and is responsible for poor prognosis of colorectal cancer. Clin. Exp. Metastasis 19:527-534. Kraemer, S.A., Meade, E.A., and DeWitt, D.L. 1992. Prostaglandin endoperoxide synthase gene structure: Identification of the transcriptional start site and 5'-flanking regulatory sequences. Arch. Biochem. Biophys. 293:391-400.
Kujubu, D.A., Fletcher, B.S., Varnum, B.C., Lim, R.W., and Herschman, H.R. 1991. TIS 10, a phorbol ester tumor promoterinducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J. Biol. Chem. 266:12866-12872.
Kutchera, W., Jones, D.A., Matsunami, N., Groden, J., Mclntyre, T.M., Zimmerman, G.A., White, R.L., and Prescott, S.M. 1996. Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: Evidence for a transcriptional effect. Proc. Natl. Acad. Sci. USA. 93:4816-4820. Leung, W.K., To, K.E, Ng, Y.E, Lee, T.L., Lau, J.Y., Chan, EK., Ng, E.K., Chung, S.C., and Sung, J.J. 2001. Association between cyclo-oxygenase-2 overexpression and missense p53 mutations in gastric cancer. Br. J. Cancer 84:335-339. Liotta, L.A., and Stetler-Stevenson, W.G. 1993. Principles of Molecular Cell Biology of Cancer: Cancer Metastasis, 4th ed. Philadelphia: J.B. Lippincott. Marusch, E, Koch, A., Schmidt, U., Zippel, R., Geissler, S., Pross, M., Roessner, A., Kockerling, E, Gastinger, I., and Lippert, H. 2002. "Colon-/rectal carcinoma" prospective studies as comprehensive surgical quality assurance. Chirurg 73:138-145. Masunaga, R., Kohno, H., Dhar, D.K., Ohno, S., Shibakita, M., Kinugasa, S., Yoshimura, H., Tachibana, M., Kubota, H., and Nagasue, N. 2000. Cyclooxygenase-2 expression correlates with tumor neovascularization and prognosis in human colorectal carcinoma patients. Clin. Cancer Res. 6:4064-4068. Milas, L. 2001. Cyclooxygenase-2 (COX-2) enzyme inhibitors as potential enhancers of tumor radioresponse. Semin. Radiat. Oncol. 11:290-299.
Ohd, J.E, Nielsen, C.K., Campbell, J., Landberg, G., Lofberg, H., and Sjolander, A. 2003. Expression of the leukotriene D4 receptor CysLT1, COX-2, and other cell survival factors in colorectal adenocarcinomas. Gastroenterology 124:57-70. Petersen, C., Petersen, S., Milas, L., Lang, EE, and Tofilon, EJ. 2000. Enhancement of intrinsic tumor cell radiosensitivity induced by a selective cyclooxygenase-2 inhibitor. Clin. Cancer Res. 6:2513-2520. Petersen, S., Thames, H.D., Nieder, C., Petersen, C., and Baumann, M. 2001. The results of colorectal cancer treatment by p53 status. Dis. Colon Rectum 44:322-334. Ratto, C., Solo, L., Ippoliti, M., Merico, M., Doglietto, G.B., and Crucitti, E 1998. Prognostic factors in colorectal cancer.
7 Role of COX2 Expression in Colorectal Cancer Literature review for clinical application. Dis. Colon Rectum 41:1033-1049. Saha, D., Datta, P.K., Sheng, H., Morrow, J.D., Wada, M., Moses, H.L., and Beauchamp, R.D. 1999. Synergistic induction of cyclooxygenase-2 by transforming growth factor- betal and epidermal growth factor inhibits apoptosis in epithelial cells. Neoplasia 1:508-517.
Sano, H., Kawahito, Y., Wilder, R.L., Hashiramoto, A., Mukai, S., Asai, K., Kimura, S., Kato, H., Kondo, M., and Hla, T. 1995. Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res. 55:3785-3789. Sato, T., Nakajima, H., Fujio, K., and Moil, Y. 1997. Enhancement of prostaglandin E2 production by epidermal growth factor requires the coordinate activation of cytosolic phospholipase A2 and cyclooxygenase 2 in human squamous carcinoma A431 cells. Prostaglandins 53:355-369. Sawaoka, H., Tsuji, S., Tsujii, M., Gunawan, E.S., Sasaki, Y., Kawano, S., and Hori, M. 1999. Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab. Invest. 79:1469-1477. Sheehan, K.M., Sheahan, K., O'Donoghue, D.P., MacSweeney, E, Conroy, R.M., Fitzgerald, D.J., and Murray, F.E. 1999. The relationship between cyclooxygenase-2 expression and colorectal cancer. JAMA 282:1254-1257. Taketo, M.M. 1998. Cyclooxygenase-2 inhibitors in tumorigenesis (Part I). J. Natl. Cancer Inst. 90:1529-1536. Tomozawa, S., Nagawa, H., Tsuno, N., Hatano, K., Osada, T., Kitayama, J., Sunami, E., Nita, M.E., Ishihara, S., Yano, H., Tsuruo, T., Shibata, Y., and Muto, T. 1999. Inhibition of
191 haematogenous metastasis of colon cancer in mice by a selective COX-2 inhibitor, JTE-522. Br. J. Cancer 81: 1274-1279. Tomozawa, S., Tsuno, N.H., Sunami, E., Hatano, K., Kitayama, J., Osada, T., Saito, S., Tsuruo, T., Shibata, Y., and Nagawa, H. 2000. Cyclooxygenase-2 overexpression correlates with tumour recurrence, especially haematogenous metastasis, of colorectal cancer. Br. J. Cancer 83:324-328. Tsujii, M., Kawano, S., and DuBois, R.N. 1997. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc. Natl. Acad. Sci. USA. 94:3336-3340. VICTOR 2000. VIOXX in colorectal cancer therapy: definition of optimal regime. CRC Trial Unit, Institute for Cancer Studies Clinical Research Block, The Medical School Edgbaston, Birmingham B 15 2TT. Wolff, H., Saukkonen, K., Anttila, S., Karjalainen, A., Vainio, H., and Ristimaki, A. 1998. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res. 58:4997-5001. Yamauchi, T., Watanabe, M., Kubota, T., Hasegawa, H., Ishii, Y., Endo, T., Kabeshima, Y., Yorozuya, K., Yamamoto, K., Mukai, M., and Kitajima, M. 2002. Cyclooxygenase-2 expression as a new marker for patients with colorectal cancer. Dis. Colon Rectum 45:98-103. Zhang, H., and Sun, X.F. 2002. Overexpression of cyclooxygenase-2 correlates with advanced stages of colorectal cancer. Am. J. Gastroenterol. 97:1037-1041.
Ziche, M., Jones, J., and Gullino, P.M. 1982. Role of prostaglandin E1 and copper in angiogenesis. J. Natl. Cancer Inst. 69:475-482.
T'
Role of Cyciooxygenase2 Expression in Colorectai Cancer Sven Petersen
Introduction Since their discovery four decades ago prostaglandins (PGs) have been assumed to be precursors for formation and growth of malignant tumors. Thus, the manipulation of PG pathways and their impact on cancer growth and treatment have become important in cancer research. The purpose of this study is to determine whether the expression pattern of cyclooxygenase2 (COX2) in a series of human rectal tumors is linked to the outcome in a well-defined cohort of patients who underwent surgery for rectal cancer.
Prostaglandins
including blood vessel tone, platelet aggregation, and immune responses. The maintenance of the gastric mucosa is regulated by PGs as well as regulation of cell growth and differentiation. PGs are involved in many pathologic conditions such as inflammatory reactions or rheumatoid disease (Kargman et al., 1995; Kutchera et al., 1996). PGs have also been implicated in cancer development with a number of tumor types found to produce more PGs than the normal tissues from which they arise (Hida et al., 1998; Kutchera et al., 1996). PGs stimulate angiogenesis and, in addition, are vasoactive agents, which may influence tumor growth and response to cytotoxic agents (Ziche et al., 1982). Therefore, tumor characteristics such as rapid growth and metastatic spread might be linked to the effect of increased intratumoral PG levels.
PGs became of interest for experimental research after they were discovered as a group of mediators in the 1960s. The precursor of all PGs is arachidonic acid, a polyunsaturated fatty acid, which is liberated from the cell membrane phospholipids by the phosp h o l i p a s e A 2. Arachidonic acid then is catalyzed by the COX enzyme to an unstable precursor PG G 2. This intermediate form is transferred to PG H 2, which is the precursor for all resulting prostaglandins including PG E 2, PG Fza, PG D 2, PG I 2, thromboxane TX A 2 and TX B 2. Prostaglandins serve as critical mediators in mammalian physiology affecting a variety of functions,
COX1 and COX2 The rate-limiting enzyme in the synthesis of PGs from arachidonic acid is COX. The PG synthase COX1 was first cloned in 1988 (Fosslien, 2000). Since 1991 it has been known that two isoforms of COX exist (Kujubu et al., 1991). COX1 is constitutively expressed in most tissues and mediates the synthesis of PGs required for normal physiologic functions. In contrast, COX2 is typically not expressed or expressed at relatively low
Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas, Volume 2: Molecular Pathology, Colorectal Carcinoma, and Prostate Carcinoma
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Copyright 9 2005 by Elsevier (USA) All rights reserved.
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levels in undisturbed healthy tissues but is inducible by an assortment of agents including proinflammatory stimuli, mitogens, and/or hormones depending on the tissue (DuBois et al., 1994; Kujubu, et al., 1991). COX1 and COX2 are encoded by genes mapping to chromosomes 9 and 1, respectively. The COX1 gene extends for 22 kilobases (kb) and includes 11 exons, producing a 2.7 kb message. COX2 is an 8.3 kb gene of 10 exons that generates a 4.3-4.5 kb message. Although the intron/exon structure of the genes is nearly identical and the encoded proteins are ~70% homologous, the regulatory elements within the genes are quite different. Whereas COX1 represents a housekeeping gene, which lacks a TATA box (Kraemer et al., 1992), the promotor of the immediate-early gene COX2 contains a TATA box and binding sites for several transcription factors including nuclear factor-~B, the nuclear factor for interleukin-6 expression and the cyclic adenosine monophosphate response element binding protein (Appleby et al., 1994). COX2 was shown to be moderately overexpressed in a large variety of tumor types including head and neck, colon, breast, and pancreatic cancers. In addition, there is considerable evidence available indicating that COX2 promotes carcinogenesis and growth of established tumors (Eberhart et al., 1994; Fujita et al., 2000; Taketo, 1998; Tsujii et al., 1997). In most tumors, COX2 expression was detected in --~40-80% of tumor cells. However, even some normal tumor cell infiltrates, particularly epithelial cells, may express COX2. Although the relationship between COX2 and tumor aggressiveness is not fully established, it seems that COX2 overexpression is related to poor patient prognosis and enhanced propensity for metastatic spread (Achiwa et al., 1999; Sheehan et al., 1999). Enhanced COX2 expression might also be related to the grade of differentiation of the tumors. For instance, in some well-differentiated adenocarcinomas of the lung an increased COX2 level was found (Wolff et al., 1988), but COX2 was also found in other forms of lung cancer (Hida et al., 1998; Khuri et al., 2001). Probably the largest number of publications deals with COX2 expression in colorectal adenomas and carcinomas, and in this case the potential clinical application of nonsteroidal anti-inflammatory drugs has been debated for decades. Most of colorectal cancer cells express COX2. The level of COX2 expression varies depending on the detection methods, and the published data suggest that according to the tumor site, COX2 expression might be more pronounced in rectal tumors compared to the colonic tumors (Dimberg et al., 1999; Sano et al., 1995). It is controversial whether COX2 expression in itself is a prognostic factor for local recurrence and/or
II Colorectal Carcinoma
survival of patients with colorectal cancer. Some authors found a significantly higher incidence of local recurrence and increased cancer-specific mortality associated with higher COX2 expression in tumor cells (Tomozawa et al., 2000), whereas others could not confirm these observations (Fujita et al., 1998). The aim of this synopsis is to determine the influence of COX2 expression in colorectal carcinoma on tumor recurrence and survival. In this context the COX2 expression in tumors of patients treated surgically for rectal cancer in our department were analyzed. The COX2 immunohistochemistry staining technique will be described in detail. Of special interest is to analyze whether there were significant differences in the group with low COX2 expression compared to the high COX2 group with reference to well-known prognostic factors (Fielding et al., 1991). In addition, our data will be compared with currently available data on COX2 expression in colorectal cancer.
COX2 Immunohistochemistry MATERIALS
1. Tissue samples embedded in paraffin. 2. For deparaffinization: 2X xylene 400 ml; isopropanol, 2X 400 ml; ethanol solution 96% in distilled water, 2X 400 ml; ethanol solution 70%, 2X 400 ml; ethanol solution 50%, 2X 400 ml; distilled water, 2X 400 ml. Phosphate buffer saline (PBS): 100 mg anhydrous calcium chloride, 200 mg monobasic potassium phosphate, 100 mg magnesium chloride, 8 g sodium chloride, and 2.16 g dibasic sodium phosphate; bring volume to 1 L with deionized distilled water (pH 7.4). 3. Solution for unmasking the antigens: citrate buffer solution: 450 ml distilled water, 9 ml of 100 mmol/L citrate, and 41 ml of 100 mmol/L sodium citrate. Tris buffer solution: 900 ml of 154 mmol/L NaC1 and 100 ml Tris-buffer stock solution. 4. For endogenous peroxidase blocking: --~400 ml of 3% hydrogen peroxide solution. 5. Incubation with primary antibody. Tris buffer solution: 900 ml of 154 mmol/L NaC1 and 100 ml Tris-buffer stock solution. Primary antibody" rabbit polyclonal antibody specific for human recombinant prostaglandin H synthase Form-2 (COX2) diluted 300-fold in antibody dilution (Dako, Code No. $3022, DakoCytomation GmbH, Hamburg, Germany). The antibody was provided by Oxford Biomedical Research Inc., Michigan. 6. For immunostaining, the Dako kit EnVision System was used. The immunostaining kit was provided by DakoCytomation GmbH. Incubation with secondary antibody alkaline phosphatase labeled polymer and
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7 Role of COX2 Expression in Colorectal Cancer substrate chromogen solution (Fast Red) was used. Distilled water, 2X 400 ml. 8. Counterstaining: hematoxylin. 9. For the dehydration of the samples, the same materials as in Step 2 were used.
METHODS Tissue samples were fixed with 4% formaldehyde in PBS, embedded in paraffin, and cut in 4 Bm thick sections. The sections were deparaffinized, hydrated through xylene and ethanol, and microwaved. The following steps were performed: 1. Rinse the samples two times in xylene for 10 min each. 2. Rinse the samples two times in isopropanol for 2-3 min each. 3. Hydrate the samples by rinsing first two times in 96% ethanol for 2-3 min each, followed by rinsing in 70% ethanol once for 2-3 min and 50% ethanol once for 2-3 min. Finally, rinse the samples two times in distilled water for 2-3 min each. 4. Microwave the samples two times for 5 min each. 5. Rinse the samples in Tris buffer two times for 5 min each. 6. Endogenous peroxidase was blocked by immersion of the samples in 3% hydrogen peroxide for 30 min at room temperature. Estimated 100 B1 peroxidase solution for each sample. 7. Rinse the samples in Tris buffer two times for 5 min each. 8. Incubation with the COX2 antibody, for this rabbit polyclonal antibody specific for human COX2 (Oxford) diluted 300-fold, was applied and sections were incubated at 4~ overnight. Estimated 100 ~tl antibody solution for each sample. 9. Rinse the samples in Tris buffer two times for 5 min each. 10. Incubation with alkaline phosphatase labeled polymer from the Dako EnVision kit for 30 min at room temperature. Estimated 100 B1 polymer solution for each sample. 11. Rinse the samples in Tris buffer two times for 5 min each. 12. Apply substrate chromogen solution (Fast Red) from the Dako EnVision kit for 8 min at room temperature. Estimated 100 B1 chromogen solution for each sample. 13. Rinse the samples in distilled water two times for 5 min each. 14. The sections were counterstained with hematoxylin (HE); incubate the samples shortly in the HE solution just until the nuclears start staining (--~5-10 seconds).
15. Rinse the samples with room temperature disfilled water until the HE staining changes color from brown to blue. 16. Dehydrate the samples by rinsing two times in distilled water for 2-3 min each. Apply 50% ethanol for 2-3 rain once and ethanol 70% for 2-3 rain one time. Rinse the samples twice for 2-3 min in 96% ethanol solution. After that, rinse the samples with isopropanol twice for 2-3 min, and finally rinse the samples twice in xylene for 10 min each. 17. Mount the samples. Nonimmunized rabbit serum was used as negative control.
Evaluation of COX2 Immunostaining The specimens immunostained for COX2 were evaluated according to the intensity and extent of positive reaction of tumor cells on a semiquantitative scale. The number of stained versus not stained epithelial cells was counted in at least 500 cancer cells in the area of the most intense staining. A labeling index of COX2 staining was calculated by dividing the number of stained cells by all counted tumor cells. The median labeling index was the base of the calculations for the Chi-square test and the univariate and multivariate analyses; patients were classified in two groups, one above and one below the median COX2 labeling index. In addition, in all tumor samples the staining intensity of COX2 was divided semiquantitatively into grades 1-4, from grades 1 (low), 2 (mild), 3 (moderate), to 4 (high). In 46 of all specimens simultaneous evaluation of the staining intensity of normal tissue mucosa was possible.
Patient Samples Specimens from 62 consecutive patients with International Union Against Cancer (UICC) stage I-III rectal cancer received radical surgical treatment at the Department of General and Abdominal Surgery at the Hospital Dresden-Friedrichstadt, in the period 19951996, were evaluated. None of the patients underwent neoadjuvant chemotherapy or radiotherapy. The mean follow-up period was 42 months (standard deviation [SD] +17 months). The tumor was curatively resected in all patients; however, no mesorectal excision was performed in general. The mean number of examined lymphnodes was 10 (SD +8).
Survival The survival of patients was quantified in two different endpoints: overall survival and disease-specific survival. The overall survival excluded all dead
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II Colorectal Carcinoma
patients independent of cause of death. The diseasespecific survival was defined as cumulative survival for patients with curative resection, excluding patients who died from cancer-related causes, with a proven local recurrence, distant metastasis, or both. Survival time was counted starting at the day of surgery.
Local Recurrence and Distant Metastasis All 62 patients had a curative resection, defined as complete removal of all macroscopically evident tumor and cancer-free resection margins on histologic examination. Patients who developed a histologically proven local recurrence, an anastomotic recurrence, or combined local recurrence with distant metastasis were included in the group of local recurrence. Distant metastasis was defined as newly discovered evidence of tumor recurrence using standard follow-up for colorectal cancer. Pulmonary metastases were detected by routine chest X-ray.
Tumor Classification and Histologic Categorization According to the World Health Organization (WHO) categorization, only adenocarcinomas (International Classification of Diseases Manual [ICDM] 8140/3 or 8480/3) were included in this study. Mucinous tumors (ICDM 8480/3) were defined as more than 50% mucin in the extracellular matrix. The tumor classification was based on the TNM (tumor, lymph nodes, metastasis) staging system, including L category for lymph vessel invasion and the V1 classification for microscopic venous invasion (Hermanek et al., 1997).
Statistical Methods Statistical analysis was performed using the SPSS 10.0.7 software package (SPSS, Inc., Chicago, IL). Actuarial survival curves were calculated and plotted according to the Kaplan-Meier life-table method. For univariate analysis, comparison between the survival curves was made using the log-rank test. Variables with p-value less than 0.05 were considered to be significant.
RESULTS AND DISCUSSION
Tumor Staining All of the 62 specimens were stained positively for COX2 with a cytoplasmic immunoreactivity. There was a typical staining pattern of granular immunoreactivity in the apical part of the epithelial
Figure 30 COX2 staining in a highly differentiated tubulopapillar rectal adenocarcinoma (G1): intensive immunoreactivity in perinuclear granular of the epithelial tumor cells, nuclei with hematoxylin counterstaining (original magnification 100X). cells (Figure 30). No staining of the nucleus was observed. This is in agreement with other publications, which also found the staining pattern predominantly localized in the cytoplasm and the nuclear envelope (Tomozawa et al., 2000; Yamauchi et al., 2002). In this study, no staining of stroma cells was observed. This finding is supported by other authors who also found no stroma staining (Yamauchi et al., 2002). In contrast, there are also publications showing COX2 staining in stroma cells (Konno et al., 2002; Tomozawa et al., 2000). The median COX2 labeling index was 0.58 (SD __.0.25). Low staining intensity was observed in 10 specimens (16%); it was mild in 18 (29%), moderate in 28 (45%), and high in 6 cases (10%).
Mucosa Staining In 46 specimens (74%), mucosa could be evaluated. In the normal tissue the COX2 staining was evaluated as high in 1 specimen (2%); the staining was moderate in 19 cases (41%), mild in 18 cases (39%), and low in 8 specimens (17 %). The distribution of staining in tumor versus normal mucosa showed no difference. There was an increase of staining intensity from the distant parts of the mucosa to the tumor-adjacent mucosal areas in 17 cases (37%). These findings are in good agreement with other studies, which found COX2 staining of adjacent mucosa only in a minority of samples
7 Role of COX2 Expression in Colorectal Cancer
(Tomozawa et al., 2000; Zhang and Sun, 2002). However, it is emphasized that COX2 is eventually detectable in normal tissue. Thus, the simple paradigm that COX2 is found only in inflammatory- and cancer-related tissues is not always true. Whether this investigation is of relevance needs to be evaluated in further studies.
COX2 as Prognostic Factor for Survival The follow-up of 62 patients with rectal carcinoma revealed that 25 patients (40.3%) died. Using the median labeling index to distinguish low from high COX2 expression for survival analysis showed that 12 patients died after a mean of 48.6 months in the group of low COX2 expression and 13 patients died in the overexpression group after 44.2 months. These results are obviously not significant in the KaplanMeier survival statistics (p = 0.69). However, using a much higher threshold of a labeling index of 0.74 for COX2 overexpression provided significant results in the survival analysis, where 13 patients in the overexpression group only had a follow-up of 34.6 months compared to 49.7 months of survival in 49 patients with low COX2 expression ( p < 0.01). In the case of colorectal cancer and survival, a large variety of potential prognostic factors are available. Besides the well-established prognostic factors, the number of biologic and molecular markers that characterize carcinomas is increasing (Ratto et al., 1998). In addition, there are surgery-related factors, such as the extent of mesorectal excision in rectal surgery, the surgeon himself, or complicating events, which affect prognosis (Hermanek, 1999a; Marusch et al., 2002).
187
Thus, Hermanek postulated the following recommendation for prognostic factors using immunohistochemistry: 1) standardization of methods to increase the acceptance of potential prognostic or predictive factors; 2) analysis of potential new markers must include the established prognostic factors with the highest statistical power (pT, pN, UICC stage, and curative resection R) (comments in Petersen et al., 2001). According to the value of prognostic factors, the Colorectal Working Group published the "American Joint Committee on Cancer Prognostic Factors Consensus Conference." In this survey, prognostic factors were classified into four groups (Compton et al., 2000). Category I includes prognostic factors that are well evaluated by published data and on which treatment strategies are based and which can modify the established TNM classification. In category IIa, factors from clinical or histologic evaluations with prognostic value might be added to the histopathologic characterization tumors. Category IIb includes well-studied prognostic factors that are not sufficiently established for category I or IIa. Prognostic variables, which are not yet established to meet criteria for category I and II, are summarized in category III. Category IV includes prognostic factors that show no consistent prognostic significance. The most important prognostic factors in category I are variables, which are included into TNM classification and the UICC stage (Compton et al., 2000; Fielding et al., 1991; Hermanek, 1999b). According to relevance of COX2 expression and survival analyses only data on immunohistochemistry (IHC) evaluation of COX2 are available (Table 4). Three studies found significant impact of COX2
Table 4 Published Data of Prognostic Influence of COX2 in Colorectal Cancer COX2 Antibody Detection (Dilution)
Cutoff (No. Patient: Negative versus Positive)
Overall Survival
DiseaseSpecific Survival
Author
Patients
Therapy
Konno et al. (2002) Masunaga et al. (2000)
56 100
IHC IHC
IBL (1:20) Cayman (1:300)
>5% (42 versus 14) >0% (24 versus 76)
Significant Significant
n.s. n.s.
13hd et al. (2003)
61
Surgery Surgery (n = 58 postoperative chemotherapy) Surgery
IHC
Cayman (1:500)
Insignificant
n.s.
Sheehan et al. (1999) Tomozawa et al.
76
Surgery
IHC
Cayman (1:500)
Low/moderate versus high/very high (n.s.) > 1% (14 versus 62)
Significant
n.s.
63
Surgery
IHC
IBL (1:40)
>50% (50 versus 13)
n.s.
Significant
232
Surgery
IHC
Alexis (1:1000)
>70% (66 versus 166) n.s.
Significant
112
n.s.
IHC
Dako (1:400)
n.s. (31 versus 81)
n.s.
(2000) Yamauchi et al. (2002) Zhang et al. (2002)
IHC, immunohistochemistry;n.s., not stated.
Insignificant
188
expression on the overall survival (Konno et al., 2002; Masunaga et al., 2000; Sheehan et al., 1999); two additional studies provided evidence that COX2 overexpression reduced the disease-specific survival (Tomozawa et al., 2000; Yamauchi et al., 2002). Their results are inconsistent with the results reported here. Using the median COX2 labeling index as a cutoff point, no significant influence on local recurrence or survival was found. The data presented here, however, are in good agreement with the results presented by Zhang et al. (2002) and (Jhd et al. (2003). In a study including 112 patients with colorectal cancer, Zhang found no impact of COX2 expression on survival (Zhang and Sun, 2002). The authors concluded that COX2 overexpression is more likely linked to cancer differentiation rather than to prognostic value. Ohd et al. also could not confirm the relevance of COX2 overexpression on overall survival in 61 patients with colorectal cancer; they only found an impact of COX2 on survival in a subgroup of UICC-stage II patients (Ohd et al., 2003). The results with IHC can vary because of different antibodies, variations in the technique of incubation and antigen fixation, subjectivity in scoring, and absence of uniform cutoff value for definition of positive tumors. Accordingly, reasons for divergent results from different publication are mainly the result of differences of the immunohistochemical staining techniques (Garewal et al., 2003). As stated in Table 4, techniques for assessing the COX2 activation vary extensively (e.g., dilution of the antibody or the incubating time). In this study the Oxford polyclonal antibody was used. In contrast, other published data on COX2 in colorectal cancer and prognostic evaluation used other antibodies. Two studies used the IBL antibody (Konno et al., 2002; Tomozawa et al., 2000); three used the Cayman antibody (Masunaga et al., 2000; Ohd et al., 2003; Sheehan et al., 1999). In one study the Dako antibody was used (Zhang and Sun, 2002); in another study the staining analysis was performed using the Alexis antibody (Yamauchi et al., 2002). Another methodologic problem is the cutoff point to distinguish COX2 overexpression. The threshold varies substantially among different studies. Masunaga et al. (2000) and Sheehan et al. (1999) considered tumors with a minimal COX2 staining as positive. In contrast, Tomozawa et al. (2000) regarded IHC as COX2 overexpressed when 50% of all tumor cells showed staining pattern. Using the median labeling index as cutoff, the data presented here provide no impact of COX2 expression on local recurrence or overall survival. In contrast, there was a significant increased number of isolated pulmonary metastasis in the COX2 overexpression group.
II C o l o r e c t a l C a r c i n o m a
Changing the cutoff point of the labeling index to 0.74, only 13 patients showed COX2 overexpression. However, in this group the disease-specific and the overall survival were significantly decreased. This result of negative impact of very high COX2 expression is in good agreement with other studies. Konno et al. (2002) and Masunaga et al. (2000) found also a decreased overall survival associated with COX2 overexpression. Tomozawa et al. (2000) and Yamauchi et al. (2002) were able to show this effect of high COX2 levels on disease-specific survival. Thus, low COX2 expression in colorectal tumors does not seem to be linked to malign potential of carcinoma. In contrast, severe COX2 overexpression indicates increased malign potency of a tumor and an increased risk of potential metastatic spread. Only a small number of data is published according to the relevance of COX2 overexpression on local recurrence of colorectal cancer (Tomozawa et al., 2000). The data presented here provide evidence that COX2 overexpression is not linked to local failure following curative resection of rectal carcinoma.
COX2 and Metastasis One of the potential reasons of decreased survival associated with COX2 overexpression is an increased capability of the overexpressing tumor for metastatic spread. The data presented here show a higher incidence of lung metastasis in the group with increased levels of COX2 expression in the tumor samples. Using the median labeling index of 0.58, isolated lung metastasis was observed in five patients; all of these metastatic events happened in the overexpression group, which was obviously statistically significant in the Kaplan-Meier analysis (p = 0.04). In eight patients the rectal carcinoma recurred locally~five times in the lower expression group and only three times in the group with COX2 overexpression. These results were insignificant ( p = 0.41). The outcome presented here gives evidence that increased levels of COX2 expression in rectal carcinoma are associated with higher potency of hematogenous metastatic spread. The latter effect of COX2 is most likely linked to higher levels of PGs that are known to cause increased metastatic events (Honn et al., 1981). Metastatic events are the result of a complicated interaction between tumor and host. Primary tumor growth is followed by tumor cell translation to the location of metastasis; this event is closely related to angiogenesis (Hejna et al., 1999; Liotta et al., 1993). Thus, increased COX2-associated neoangionenesis might explain higher rates of hemateneous metastasis
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7 Role of COX2 Expression in Colorectal Cancer in tumors with COX2 overexpression (Cianchi et al., 2001; Costa et al., 2002; Gallo et al., 2001). Cianchi et al. (2001) reported a strong correlation between COX2 and vascular endothelial growth factor (VEGF) expression in human colorectal cancer specimens, and Gallo et al. (2001) showed head and neck tumors with high levels of VEGF and COX2 expression and significantly higher vascularization. Tomozawa et al. (2000) also reported increased rates of hematogenous metastasis in patients with COX2 overexpression in the tumor samples. These data were supported by experimental results from the same group. In a mouse model, they evaluated the number of lung metastases following intravenous tumor injection. The number of pulmonary metastases was significantly reduced by application of selective COX2 inhibitor (Tomozawa et al., 1999). The biologic background of the influence of COX2 expression on tumor growth and metastatic potential is a focus of investigation. There is evidence that high COX2 expression is associated with mutant p53 (Leung et al., 2001) or with EGF and nuclear factor-rJ3 expression (Saha et al., 1999; Sato et al., 1997). In addition, data are available from cell culture and in vivo studies indicating that elevated levels of COX2 promote angiogenesis, which might influence the metastatic behavior of tumor cells (Kishi et al., 2000; Sawaoka et al., 1999). Another mechanism that might be involved in COX2-associated metastatic spread is the connection of COX2 and the matrix-metalloproteinase 2 (MMP-2). MMP-2 modulates cell surface integrity and was shown to be associated with increased metastatic spread in a variety of tumors (Baker et al., 2000; Barozzi et al., 2002). Using COX2 transfected colorectal cancer cell in in vitro experiments it was shown that COX2 induced increased MMP-2 activity (Tsujii et al., 1997). Other metastasis-inducing factors that might be linked to COX2 overexpression are urokinase-type plasminogen activator (uPA) and interleukins (Fosslien, 2000; Konno et al., 2002). Because there is increasing emphasis to stratify treatment modalities according to molecular parameters, the critical evaluation of prognostic molecular factors has become essential (Petersen et al., 2001). According to COX2 overexpression in tumor samples, and the data presented here, the conclusion can be drawn that COX2 overexpression is linked to increased tumor aggressiveness. However, the immunohistochemical detection of COX2 is not yet an established prognostic factor. According to the American Joint Committee on Cancer Prognostic Factors Consensus Conference, at present COX2 overexpression can be classified as level IIb to III prognostic factor (Compton et al., 2000).
CONCLUSIONS The data presented here indicate that COX2 overexpression, detected with IHC analysis, has a marginal impact on the survival of surgically treated patients with colorectal cancer, but there is need for standardization of the technical approach to assess COX2 overexpression. Presently, there is not enough evidence that COX2 mutation is a predictive factor for response to adjuvant treatment. One of the major questions is how to treat patients with an immunohistochemical COX2 overexpression, cannot be answered sufficiently from the data. The role of COX2 as a predictive factor in colorectal cancer, therefore, needs to be addressed in appropriate clinical trials. Since selective COX2 inhibitors became available, the detection of COX2 expression has played an emerging role in the stratification of treatment with COX2 inhibitors in human malignant tumors. Experimental data show that COX2 inhibition, especially in combination with other treatment modalities, might be one part of cancer treatment in the future (Milas, 2001; Petersen et al., 2000). Studies are ongoing to evaluate the role of COX2 inhibition in human malignant neoplasms (Chang, 2002; VICTOR, 2000). The results of these studies will soon give evidence whether COX2 needs to be focused more in cancer treatment.
Acknowledgments I gratefully acknowledge the excellent technical assistance of Gunter Haroske and Mrs. Blumentritt from the Department of Pathology; Georg Schmorl, General Hospital DresdenFriedrichstadt; and Cordula Petersen and Michael Baumann from the Department of Radiotherapy and Oncology, University Dresden, Germany, in the immunohistochemistry studies.
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