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A Prognostic Marker in Colon Cancer

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A Prognostic Marker in Colon Cancer ANTICANCER RESEARCH 32: 3869-3874 (2012) The Expression of CYP2W1: A Prognostic Marker in Colon Cancer KRISTINA STENSTEDT1,4, MARIA HALLSTROM3, INGER JOHANSSON2, MAGNUS INGELMAN-SUNDBERG2, PETER RAGNHAMMAR3 and DAVID EDLER1,4 Departments of 1Molecular Medicine and Surgery, 2Physiology and Pharmacology, and 3Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden; 4Centre for Surgical Gastroenterology, Karolinska University Hospital, Stockholm, Sweden Abstract. Aim: The enzyme Cytochrome P450 2W1 has been shown to catalytically activate compounds to (CYP2W1) is found in fetal colon tissue and is also detected cytotoxic products, the enzyme might be used as a novel drug in colorectal cancer but not in non-transformed tissue. In a target for the treatment of colon cancer. pilot study, we reported that the immunohistochemically- detected expression of CYP2W1 might be of prognostic value P450 cytochromes (CYPs) belong to a superfamily of heme- since high expression of CYP2W1 was indicative of a worse containing enzymes which play important roles in prognosis. The aim of this study was to validate the pilot biosynthesis and metabolism of endogenous compounds, as study’s results using a larger, independent group of patients well as in the metabolism and detoxification of various with colon cancer. Materials and Methods: exogenous compounds, such as drugs, carcinogens and toxic Immunohistochemical detection of CYP2W1 in 235 environmental agents. Pro-carcinogens can also be activated malignant colon tumors of stage II and III, was carried out to carcinogens by CYPs. using a polyclonal antibody. Grading of staining was carried There are 57 CYP isoenzymes found in humans and they out by two independent readers. The highest grade that are arranged into subgroups based on their similarities in involved more than 5% of the tumor area on each slide was amino acid sequences. Most CYPs have well-known metabolic used for the classification of CYP2W1 expression. Results: functions, while others currently have no assigned function. CYP2W1 was expressed at high levels in 30% of the tumors. Several CYP enzymes are expressed in human tumors (1-4). In the entire colon cancer group it was an independent CYPs can act as bioactivators and pro-drugs such as the novel prognostic factor in multivariate analysis (p=0.04), where agent 1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8- high expression (grade 3) correlated with worse outcome. dihydroxyanthracene-9,10-dion (AQ4N) can be activated in CYP2W1 expression was an independent prognostic factor in tumors expressing the enzyme (5). Such challenging targeted the subgroup of patients with colon cancer stage III therapy could be possible if the enzyme is a tumor-specific (p=0.003), but not for those with stage II. In 107 cases, two enzyme with no expression in normal tissue. slices from different areas of the same tumor were available, CYP2W1 has been cloned and expressed. The human and no significant difference in CYP2W1 expression between CYP2W1 gene is located on chromosome 7p22.3. The function the slices was observed (r=0.53, p<0.001). Conclusion: The of CYP2W1 is not fully understood, although recent studies results of the current study were in agreement with those of have shown that CYP2W1 can catalyze various chemical the previous pilot study and show that higher expression of reactions, e.g. benzphetamine N-demethylation, arachidonic CYP2W1 seems to be of prognostic value in colon cancer. acid oxidation, and metabolism of indole and 3-methylindole Furthermore, we found equal expression in slices from two (6). The enzyme is partly glycosylated in human embryonic different areas of the same tumor. Since the CYP2W1 enzyme kidney (HEK) 293 cells and colon tumors, and it mainly has a reverse orientation in the endoplasmic reticulum (ER), compared to other CYPs. The significance of this reversed topology is not yet understood and it is, as far as we know, the Correspondence to: Kristina Stenstedt, Centre for Surgical only CYP oriented this way in the cell (6). Despite this fact Gastroenterology, P9:03, Karolinska University Hospital, 171 76 the enzyme may activate aflatoxin B1, for example, to Stockholm, Sweden. E-mail: [email protected] cytotoxic products and metabolize indolines (6). Key Words: Cytochrome P450, CYP2W1, colonic cancer, prognostic The CYP2W1 protein is found in rat fetal colon but not in marker. any other tissues of adult rat. CYP2W1 is expressed in 0250-7005/2012 $2.00+.40 3869 ANTICANCER RESEARCH 32: 3869-3874 (2012) HepG2 human hepatoma cell line and in some transformed Table I. Patients’ characteristics in the group of 235 patients with tissues (7). CYP2W1 expression is high in gastrointestinal colonic cancer and CYP2W1 expression. tumors, especially in colonic and rectal tumors (8). Analysis Number CYP2W1 Chi- of mRNA levels in colonic tumors showed that 60% of the of expression square tumors had overexpression of mRNA compared to HepG2 patients 0-2 vs. 3 p-value cells. The protein levels detected with western blotting showed that the colonic tumors could be divided into two phenotypes: those with high CYP2W1 expression and those Total 235 164 (70%) vs. 71 (30%) Gender 0.7 with low expression (8). Male 127 (54%) 90 (71%) vs. 37 (29%) A polyclonal antibody against a CYP2W1 15-amino acid Female 108 (46%) 74 (69%) vs. 34 (31%) C-terminal peptide has been produced and described (7). Our Age 0.02 group performed a pilot study including 162 patients with <66 years 114 (49%) 88 (77%) vs. 26 (23%) colorectal cancer stages II and III to detect the intratumoral ≥66 years 121 (51%) 76 (63%) vs. 45 (37%) Stage 0.7 CYP2W1 expression and to assess whether high expression, II 103 (44%) 73 (71%) vs. 30 (29%) detected by immunohistochemistry, correlated with prognosis. III 132 (56%) 91 (69%) vs. 41 (31%) That study showed that expression of the enzyme was an Site 0.8 independent prognostic factor for overall survival in the entire Right colon 108 (46%) 77 (71%) vs. 31 (29%) group, where a high expression was associated with worse Transverse colon 27 (11%) 17 (63%) vs. 10 (37%) Descending colon 19 (8%) 12 (63%) vs. 7 (37%) clinical outcome (9). When analyzing the colonic cancer and Sigmoid 78 (33%) 55 (71%) vs. 23 (29%) rectal cancer groups separately, CYP2W1 was an independent Unknown 3 (2%) prognostic factor in colonic cancer but not in rectal cancer. Grade (differentiation) 0.13 The present investigation was carried out in order to Low 58 (25%) 46 (79%) vs. 12 (21%) validate this preliminary finding. Since the result was most Medium 159 (67%) 105 (66%) vs. 54 (34%) High 14 (6%) 11 (79%) vs. 3 (21%) obvious in the colonic cancer group, we chose to analyze Unknown 4 (2%) another, twice as large, independent group of patients with Treatment 0.9 colonic cancer from the same cohort with the same method Surgery alone 125 (53%) 87 (70%) vs. 38 (30%) as the one used in the previous study. We also addressed the Surgery + adjuvant 110 (47%) 77 (70%) vs. 33 (30%) question of homogeneity in CYP2W1 expression, whether or Number of nodes examined 0.6 <12 analyzed nodes 139 (59%) 98 (70%) vs. 41 (30%) not the expression was equal in slices from two different ≥12 analyzed nodes 17 (7%) 13 (76%) vs. 4 (24%) areas of the same tumor. Unknown 79 (34%) Materials and Methods Patients. The primary tumors of 235 patients with colonic cancer of stages II and III from 20 different Swedish hospitals were examined 4-μm thick sections. Immunohistochemical analysis of CYP2W1 with respect to CYP2W1 expression. The surgical specimens were expression was performed using the avidin-biotin-peroxidase complex derived from adjuvant Nordic trials where patients up to the age of technique (Vectastain® Rabbit IgG ABC-kit, Vector Labs, 75 years, with radically-resected colorectal cancer of stages II and Burlingame, California, USA) and the CYP2W1 polyclonal antibody. III were included. Parameters of clinical outcome were obtained The tumor slides were de-paraffinized in xylene and rehydrated in from the Regional Oncological Centers. The patients’ demographics ethanol and thereafter incubated in a 3% hydrogen peroxide to inhibit and tumor characteristics are listed in Table I. All patients were the endogenous peroxidase activity. In order to reduce non-specific randomized to surgery alone or surgery followed by adjuvant background staining, the slides were blocked with goat serum for 30 chemotherapy. The adjuvant chemotherapy regimens included 5- min followed by incubation with the CYP2W1 antibody, at 4˚C fluorouracil (5-FU)/levamisole for 12 months or 5-FU/leucovorin overnight. The antibody was used at a dilution of 1:1000. The samples for 4-5 months according to either a modified Mayo Clinic schedule were then rinsed and incubated with biotinylated secondary antibodies or a Nordic schedule. Some centers also randomized patients treated and thereafter rinsed and incubated with avidin-biotin-peroxidase with 5-FU/leucovorin to receive or not levamisole (9). Adjuvant complexes. Visualization of immunostaining was achieved by therapy was initiated within 11 weeks after surgery. immersion of slides in 0.05% 3,3’-diaminobenzidine For 107 patients, we had access to two slices from different areas tetrahydrochloride, followed by counterstaining with hematoxylin. of the same tumor. These were compared in order to assess homogeneity regarding the expression pattern. Evaluation of immunohistochemistry. CYP2W1 staining intensity The study was approved by the local Ethical Committee at the was defined by a visual grading scale from 0 to 3 (grade 0=no Karolinska Institutet. staining, grade 1=weak, grade 2=moderate, grade 3=intense staining). Each time a set of tumor samples was stained, reference Immunohistochemical analysis. The examined colorectal specimens slices were included as well, as one negative control slice incubated were derived from formalin-fixated, paraffin-embedded tumors in with pre-immune serum.
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