Yonago Acta medica 2007;50:81–87

Topoisomerase I Protein Expression and Prognosis ofPatients with Colorectal Cancer
Masayuki Ataka, Masahide Ikeguchi, Manabu Yamamoto, Masashi Inoue, Takashi
Tanida, Shin-ichi Oka and Kuniyuki Katano
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504 Japan

Topoisomerase I (Topo I) is known as a target for chemotherapy in advanced or recurrent colorectal cancer. In order to prolong the survival of patients with colorectal cancer
or to prevent ineffective chemotherapy, we evaluated clinicopathological characteristics
of Topo I protein in colorectal cancer. Also, we estimated whether Topo I protein expression of primary tumors could be a parameter for chemosensitivity of Topo I inhibitor in patients with cancer recurrence. Immunohistochemical detection of Topo I protein was performed in 104 surgically obtained specimens. Topo I protein was detected
in 45 of 104 patients (43.2%). Topo I protein expression closely correlated with tumor
progression, histpathological differentiation and poor prognosis of patients. Sixteen
patients with recurrent cancer had been treated with Topo I inhibitor. Topo I inhibitor significantly prolonged the survival of 12 patients who had Topo I-positive primarytumors. Topo I protein expression in colorectal cancer may be a biological marker forchemosensitivity of tumors against Topo I inhibitors.
Key words: chemosensitivity; colorectal cancer; DNA topoisomerase I; immunohistochemistry;
prognosis

DNA topoisomerase I (Topo I) belongs to theDNA topoisomerase multimember family, whichis essential for DNA topology modulation. TopoI transiently cleaves one strand of DNA, allowing
relaxation of the supercoiled DNA. This process
is important in cell replication, translation, recombination and repair (Gupta et al., 1995). ThroughWestern and Northern blotting, Topo I protein
and mRNA level were found to be more abundantin several human tumors than in normal tissues(Husain et al., 1994; Rowinsky et al., 1994; vander Zee et al., 1994; Giaccone et al., 1995).
Topo I is also a target for anticancer drugs,
camptothecin and its derivatives (O’Leary andMuggia, 1998). Topo I-inhibiting drugs interferewith Topo I function by binding to Topo I at its
active site, and prevent re-ligation of the DNA
strand (Goldwassew et al., 1995). Camptothecin
inhibits Topo I by forming stable Topo I–DNA
cleavage complexes, and is specifically cytotoxicfor S-phase cells (Hsiang et al., 1989). In vitro,
tumor cells with a high level of Topo I protein
respond better to Topo I inhibitors (Staley et al.,1999).
CPT-11, a derivative of camptothecin, has beenused as one of the key drugs for treating colorectal cancer (Paradiso et al., 2004; Vallböhmer etal., 2006). In the last few years, it has been shownthat when used in combination with 5-fluorouracil and leucovorin, both CPT-11 and oxaliplatin

Abbreviation: Topo I, topoisomerase I81

M. Ataka et al.

treatments demonstrated significant improvementin the clinical outcome of patients with advanced
colorectal cancer (Doillard et al., 2000; de Gramont et al., 2000). However, response rates forthese chemotherapeutic regimens still remain
about 40% to 50%. In the present study, we investigated the clinicopathological characteristics
of colorectal tumors with Topo I protein expression. Also, we analyzed Topo I protein expression
in primary tumors could be a biomarker of chemosensitivity for recurrent patients or not.

peroxide chromogen substrate for 10 min at room
temperature, washed in running water for 2 to3 min, counterstained in Mayer’s haematoxylin.
Normal tonsil tissue was used as a positive control. It is known that normal tonsil tissue is wellstained by immunohistochemistry which uses
Topo I antibody (Rasheed and Rubin, 2003).
Scoring system
Tumor cells expressing Topo I immunoreactivity
were quantified by 2 independent observers whoevaluated at least 1,000 neoplastic cells in consecutive areas of neoplastic tissues. If there wasa Topo I-positive cell in the tumor, the sample
was classified as “positive”. If there were no TopoI-positive cells, the sample was classified as “negative” (Paradiso et al., 2004).

Materials and Methods
Tumor samples
We obtained tumors and non-cancerous normal mucosa from 104 patients who underwent
colorectal resection between 1992 and 2001.
Samples were collected immediately after surgical resection of specimens. The tissues werefixed in 10% buffered formalin and embedded inparaffin. Four micrometer histological sections
were destined to specific immunohistochemicaldeterminations.

Patients
Clinicopathological findings of colorectal cancer
were defined according to Dukes’ classification(Dukes and Bussey, 1958). In all 104 patients,
curative colorectal resection was performed between 1992 and 2001 at Tottori University Hospital. Patients agreed to the use of their tissues byinformed consent. None of the patients received
preoperative chemotherapy, and all were followed
until December 2006. The types of cancer recurrence were established by computed tomography,
performed at least twice a year.

Immunohistochemistry
After paraffin-embedded sections on the slides
were dewaxed and rehydrated gradually withgraded alcohols, antigen retrieval was performedby autoclaving in 10 mol citrate buffer for 30 min.
Endogenous peroxidase activity was blocked bymethanol and 0.3% hydrogen peroxide. Slides
were then incubated with primary anti Topo I
monoclonal antibody (Clone 1D6, 1:50 dilution,
Novocastra Laboratories, Newcastle, UnitedKingdom) for 1 h at room temperature. After incubation, the specimens were washed twice withphosphate-buffered saline solution (pH 7.6) andprocessed with the streptavidin-biotin peroxidase
method according to the manufacturer’s recommendations. The slides were then incubated in diaminobenzidine tetrahydrochloride and hydrogen-Statistical analysis
The chi-square test was used to compare thedifferences between the 2 groups. The overall
and disease-free survivals were estimated using
Kaplan-Meier’s method and compared using a2-sided log rank test. Cox’s proportional hazards
regression model was used to estimate the predictive power of Topo I protein expression on clinicaloutcome. Two-sided tests were computed, and P <0.05 was considered statistically significant.

82Topo I expression in colorectal cancer

ABFig. 1. Immunohistochemical topoisomerase I protein expression in normal colorectal mucosa adjacent to tumor (A)and in normal skin (B). Bar = 200 μm.

Resultsin normal cells in basal layer of the skin adjacent
to rectal cancer of patiens who had undergone
amputation of the rectum (Fig. 1). Topo I immunostaining was mainly located in the nucleus ofcancer cells (Fig. 2).

Topo I expression in tumors and in noncancerous tissues
Topo I protein expression was not detected innormal colorectal mucosa. But it was detected

Fig. 2. Immunohistochemical topoisomerase I protein expression in advanced colorectal cancer. Strong nuclear expression of the protein is noted. Bar = 200 μm.

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