A total of 175 patients (99 men, 76 women) with a mean age of 67 years (range 12–91 years) who underwent surgery for colorectal cancer from November 2000 to October 2006 at Mie University Hospital, Japan, were enrolled in the study. Fresh frozen surgical cancer samples, for which complete clinical data and isolated RNA of sufficient quality for real-time PCR were available, were obtained from the patients. No patients had received anticancer therapy prior to surgery and there were no perioperative mortalities among these patients.

The locations of tumors and distant metastases were determined by barium enema, colonoscopy, computerized tomography (CT) and magnetic resonance imaging (MRI). The location of the primary lesion was in the rectum in 57 patients, the sigmoid colon in 63 patients, the ascending colon in 38 patients, the transverse colon in 13 patients and the descending colon in 4 patients. Forty-one of the 175 patients had synchronous distant metastasis (liver, lung and peritoneum) at the time of surgery. Resection of the primary tumor was performed in all patients and simultaneous partial hepatectomy for liver metastasis was performed in 9 of these 27 patients. Only 16 patients had poorly differentiated adenocarcinoma, whereas 159 patients had either well or moderately differentiated adenocarcinoma. All patients were classified according to UICC staging of resected specimens. Overall, 39 patients had UICC stage I (T1–2N0M0) disease, 51 patients had UICC stage II (T3–4N0M0) disease, 44 patients had UICC stage III (TXN1–2M0) disease and 41 patients had UICC stage IV (TXNXM1) disease. Stage III and IV patients received fluorouracil-based chemotherapy, whereas no adjuvant therapy was given to stage I or II patients. Patients were observed at 3-month intervals for 24 months after surgery, then every 6 months for 3 years and then on an annual basis. A history was obtained and a physical examination was performed at each visit. Chest X-rays, colonoscopies and CTs were performed annually. The median follow-up time was 43.2 months (mean, 43.2±29.8 months).

Among the 175 patients evaluated, 40 patients died due to primary or recurrent disease. Matched control samples were acquired from adjacent normal mucosa located far from the tumor site. These samples were frozen in liquid nitrogen immediately after surgical resection and were stored at −80ºC until RNA extraction. The purpose of this study was to analyze KiSS1 expression made post hoc using previously and prospectively collected tissue samples. Written informed consent was obtained from all patients. The diagnosis of colorectal cancer was confirmed in all 175 patients on the basis of clinicopathological findings.

Tumor specimens were homogenized with a Mixer Mill MM 300 homogenizer (Qiagen, Chatsworth, CA, USA). Total RNA was isolated using an RNeasy Mini kit (Qiagen), used according to the manufacturer’s instructions. cDNA was synthesized from 5.0 mg of RNA with random hexamer primers and Superscript™ III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) used according to the manufacturer’s instructions.

Quantitative PCR (qPCR) analysis was performed using the TaqMan^® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The relative abundance of target transcripts, measured using TaqMan^® probes for KiSS1 (Hs00158486_m1, TaqMan^® Gene Expression Assays; Applied Biosystems) was normalized to the expression level of β-actin (Hs99999903_m1, TaqMan^® Gene Expression Assays; Applied Biosystems) and measured using Applied Biosystems StepOne™ Software v2.1. All reactions for standard samples and for patient samples were performed in triplicate. The data were averaged from the values obtained in each reaction.

Immunohistochemical analysis of KiSS1 protein expression was performed on colorectal cancer surgical specimens using avidin-biotin peroxidase methods (DakoCytomation, Carpinteria, CA, USA) on formalin-fixed, paraffin-embedded tissues. All sections were counterstained with hematoxylin. A primary rabbit polyclonal antibody against KiSS1 (sc101246; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used at a dilution of 1:50.

Statistical analysis was performed using StatView software (version 5; Abacus Concepts, Inc., Berkeley, CA, USA). Results are expressed as the means ± standard deviation (SD) and differences were evaluated by the Wilcoxon rank correlations test. Mann-Whitney U tests were used to evaluate differences between unpaired observations. Analyses of nonparametric receiver operating characteristics (ROCs) were performed to calculate the cut-off values according to the most accurate value obtained using MedCalc 7.2 for Windows (MedCalc, Mariakerke, Belgium). Actuarial survival curves were obtained using the Kaplan-Meier method and comparisons were made using log-rank tests. The Cox proportional hazards regression model was used for multivariate analysis after the relevant prognostic variables had been defined by univariate analysis. Logistic regression analysis was used to evaluate the independent influence of factors on peritoneal dissemination as the final outcome. Two-sided P-values <0.05 were considered to indicate statistically significant differences.

Expression values (relative mRNA levels) of KiSS1 were expressed as ratios between the gene of interest (KiSS1) and an internal reference gene (β-actin), which provided a normalization factor for the amount of RNA. In clinical samples obtained from patients with colorectal cancer, quantitative real-time RT-PCR revealed that the mean KiSS1 mRNA expression level in cancerous tissues was significantly higher than that in corresponding adjacent normal mucosa (cancerous tissue, 373.2±1129.6; adjacent normal mucosa, 0.06±0.12; P<0.001) (Fig. 1A). In addition, the mean level of KiSS1 expression was likely to decrease in a stage-dependent manner (Fig. 1B).

Table I shows clinicopathological variables and KiSS1 mRNA expression levels in tumor specimens from colorectal cancer patients. The KiSS1 expression level was significantly associated with lymph node metastasis. In the entire colorectal cancer population, the best pair of values for highest sensitivity (58.5%) and highest specificity (61.9%) was found using a peak KiSS1 mRNA expression cut-off point of 9.276. Patients with KiSS1 expression levels <9.276 in cancerous tissues were assigned to the low-expression group (n=75), whereas those with values ≥9.276 were assigned to the high-expression group (n=100). Patients in the low-expression group had significantly poorer prognoses than those in the high-expression group (log-rank test, P=0.014) (Fig. 2).

The association between KiSS1 expression and prognosis in colorectal cancer patients was evaluated by multivariate analysis. Table II shows the resulting risk ratios and 95% confidence intervals (CIs) calculated using the Cox proportional hazards model. Based on a Cox univariate proportional hazards analysis, advanced T classification (T3, T4), venous invasion, lymph node metastasis and low KiSS1 expression were associated with poor prognosis. Our multivariate analysis revealed that advanced T classification, lymph node metastasis and low KiSS1 expression were independent prognostic factors for colorectal cancer patients. Factors that correlated with lymph node metastasis were analyzed by logistic regression analysis (Table III). Notably, T classification and low KiSS1 expression were independent factors for lymph node metastasis in colorectal cancer patients.

Immunohistochemical analysis revealed that the KiSS1 protein was predominantly expressed in the cytoplasm of primary colorectal cancer cells without expression in normal mucosa (Fig. 3A). Although KiSS1 protein was highly expressed in the early stage of colorectal cancer cells (Fig. 3B), KiSS1 expression was decreased in advanced-stage colorectal cancer tissues (Fig. 3C).