SUPPLEMENTARY METHODS
Patient eligibility and treatment plan
Between 1998 and 2004, a total of 172 patients with rectal cancer, confirmed as having an adenocarcinoma of the rectum by using a colon-scopic biopsy, were enrolled for this study (Table-1). All patients were regularly monitored after diagnosis until death or their last appointment at our hospital. The institutional review board had approved procurement of formalin-fixed tissue of locally advanced rectal cancer patients for this study (IRB 10009-L04). By computed tomograph (CT), and pelvic magnetic resonance imaging (MRI) findings, ore-treatment tumor (Pre-T) stage was recorded. Pre-treatment node (Pre-N) stages were also decided on the basis of CT, and MRI findings. Enlarged (≥ 10 mm) lymph nodes being seen on any preoperative imaging examinations are defined as Pre-N (+). Distant metastasis was excluded in all cases if noted by chest X-radiography and abdominopelvic CT.
Pre-operatively, radiotherapy with a total dose of 45 Gy in 25 fractions was given over a period of five weeks with concurrent 24-hours continuous infusion of 5-FU. Radiotherapy with standard 4-field box technique was applied and clinical target volume included primary tumor as well as lymph nodes including perirectal, pre-sacral, hypogastric, obturator, and iliac. Four weeks after completion of neoadjuvant therapy,low abdominal resection (LAR) or abdomino-perineal resection (APR) was operated. If the pre- or post-treatment stage of the tumor was beyond T3 or N1, adjuvant systemic chemotherapy was indicated. The regimens were as follows: intravenous bolus of 5-FU 370 mg/m2/day and leucovorin 20 mg/m2/day on days 1–5 every 28 days for 6 courses. Postoperative follow-ups on all patients were conducted every three months for three years and clinical examination was performed during each follow-up. After three years, patients underwent follow-up examinations every six months. Measurement of serum carcinoembryonic antigen (CEA) levels, abdomen sonography and chest X-rays were arranged every six months for 5 years. Abdominopelvic CT was taken every six months for 3 years. All patients were regularly monitored after diagnosis until death or their last appointment by the surgeons, medical oncologists, and radiation oncologists at our hospital and the mean follow-up duration is 48.2 months (range, 6.2 to 131.2).
Histopathologic evaluation and SKP2 immunohistochemistry
After surgery, two pathologists (TJC and HYH) who had not been informed of the patient’s clinical information analyzed the tumor specimens. Tissue sections from initial tumour biopsies were cut onto precoated slides from paraffin-embedded tissue blocks at 3 mm thickness. Post-treatment T and N stages of all patients were documented staged according to the 7th American Joint Committee on Cancer (AJCC) TNM staging system [17]. Tumor regression grade (TRG), as described by Dworak et al [21], was used as end-points for evaluation of tumor response after CRT. The characteristics of each grade were following: grade 0, no regression; grade 1, minor regression (dominant tumor mass with obvious fibrosis in 25% or less of the tumor mass); grade 2, moderate regression (dominant tumor mass with obvious fibrosis in 26% to 50% of the tumor mass); grade 3, good regression (dominant fibrosis outgrowing the tumor mass; ie, more than 50% tumor regression); and grade 4, total regression (no viable tumor cells, only fibrotic mass).
For SKP2 immunostaining, the slides were dewaxed in xylene and rehydrated through graded alcohols to water. For antigen retrieval, slides were pressure cooked in 10 mmol/L citrate buffer (pH 6) for 7 minutes. The slides were then washed using TBS buffer with 0.1% Tween 80 for this and subsequent washes. Endogenous peroxidase activity was quenched by 3% H2O2 treatment. After washing, the slides were incubated for 1 hour at room temperature with primary antibodies targeting SKP2 (2C8D9, 1:100; Zymed, San Francisco, CA). Primary antibodies were detected using the ChemMate DAKO EnVision kit (DAKO, K5001). The slides were incubated with the secondary antibody for 30 minutes and developed with 3,3-diaminobenzidine for 5 minutes. Slides were then counterstained with Gill’s hematoxylin. Coverslips were applied with CureMount mounting medium (instrumedicus). Incubation without the primary antibody was used as a negative control. By using a multiheaded microscope to reach a consensus for each case, immunoexpression of SKP2, was scored by two pathologists (TJC and HYH) without prior knowledge of clinical results. Five groups of various expression levels from 0 to 4+, denoting none, 1%~24%, 25%~49%, 50%~74% and 75%~100% of tumour cells with moderate to strong nuclear reactivity were clarified according to the percentage of tumour cells with SKP2 immunoexpression for each specimen.