Eligible patients had pathologically confirmed clinically resectable stage T3-T4 and N0-N2 adenocarcinoma of the distal rectum with no distant metastases. Entry criteria were: inferior margin within 15 cm of the anal verge and palpable on digital examination; World Health Organization (WHO) performance status 0-2; age 18-80 years; adequate hematologic (neutrophils >1500/mm³), hepatic (bilirubin <2 upper normal limit), and renal (creatinine <150 μmol/L) function. Patients with prior malignancies (excluding localized epithelial skin or cervical cancer), chemotherapy, and/or pelvic radiotherapy were excluded. The study was approved by local ethics committees and was conducted according to the Declaration of Helsinki. Written informed consent was obtained from each patient before enrollment.

Pretreatment evaluation included a complete medical history and physical examination (including digital rectal examination), complete blood count and chemistry profile, electrocardiogram, detection of DPD activity (requiring a 10 days period), endorectal ultrasound (EUS), endoscopy with biopsy, chest X-ray, and abdomen-pelvis computed tomography (CT) scan.

Chemotherapy consisted of UFT 300 mg/m²/day and LV 75 mg/day orally in three divided doses taken at approximately 8-hour intervals (either 1 hour before or at least 1 hour after food), 5 days/week for 5 weeks (days 1-33). Pelvic radiotherapy (daily dose 1.8 Gy), delivered concurrently with chemotherapy, was delivered using a conventional four-field box technique (anterior and posterior fields, plus right and left lateral fields) and with a minimum of 10 megavoltage photons. A total dose of 45 Gy was delivered to the isocenter of the four fields. The planning target volume was defined as the clinical target volume, i.e. the primary tumor, iliac lymph nodes, and mesorectum in toto, plus a 10-15 mm margin. Diagnostic imaging was used to define the gross target volume.

Surgical resection was performed 4-6 weeks after completion of chemoradiotherapy using a standardized technique with mesorectal excision. Postoperative chemotherapy could be prescribed for patients who were lymph-node positive and who responded to preoperative treatment. These patients received 5 cycles of the same UFT with LV regimen or 5-FU/LV (de Gramont or Mayo clinic regimens) 3-6 weeks after surgery.

Toxicity, graded using WHO criteria for chemotherapy and early radiotherapy toxicity, was assessed at weekly clinic and biologic examinations during chemoradiotherapy and at the end of treatment 19 (Miller et al, 1981). Late radiotherapy toxicity was graded according to the SOMA-LENT criteria 20 (Mornex et al, 1997). In the event of grade 2-4 adverse events, chemotherapy was withheld until evidence of hematologic recovery (neutrophils ≥ 1500/mm³ and platelets ≥100 000/mm³) and complete resolution of all nonhematologic adverse events (other than alopecia) to baseline or grade 1. The UFT dose was reduced by one capsule per day if grade 3/4 toxicity occurred. Radiotherapy was postponed for 1-2 weeks in the event of grade 3 diarrhea that persisted despite UFT discontinuation; radiotherapy was stopped if the grade 3/4 diarrhea persisted for longer than 2 weeks.

EUS, chest X-ray, and CT scan were performed at baseline (within 15 days before chemoradiotherapy) and after the end of chemoradiotherapy (within 7 days before surgery). Echoendoscopic and pathologic stages were defined using the tumor node metastasis (TNM) staging system (version 5.0) 21 (Sobin and Wittekind, 1997). Initially, it was planned to determine tumor downstaging by comparing posttreatment ultrasound (u)TNM stage (EUS) with the preoperative uTNM stage. However, due to the limitations of preoperative EUS because of inflammatory effects and tissue modifications and the limited number of assessable patients, we focused on pathological results and compared posttreatment pathological (p)TNM stage to preoperative uTNM stage. Primary tumor and node downstaging were defined as reductions in T and N stages by at least one level. No response was defined as similar pTNM and uTNM classifications. A pCR was defined as the absence of any residual tumor cells in the operative specimen 22 (Meterissian et al, 1994). Lymph nodes were examined after treatment with a clarification solution in order to aid detection and improve the reliability of the pathologic N staging. All resected specimens from the different hospitals were centralized and reexamined in a reference pathology laboratory for a central review.

Sphincter function was scored according to the Memorial Sloan Kettering Cancer Center scale 23 (Minsky et al, 1995). Progression-free survival (PFS) and overall survival (OS) were measured from the time of inclusion to the time of disease progression and death (or last follow-up), respectively.

DPYD genotyping included examination of 24 relevant gene variants potentially involved in DPD deficiency. Single nucleotide polymorphisms (SNPs) were detected using a real-time mini-sequencing method 24 (Morel et al, 2005). Dihydrouracil and uracil plasma concentrations were determined simultaneously using a liquid chromatography method 25 (Remaud et al, 2005); if DPD deficiency was suspected, the plasma dihydrouracil:uracil (UH₂:U) ratio was determined 26 (Boisdron-Celle et al, 2007). UH₂:U ratios were assessed as previously described 27 (Gamelin et al, 1999). When DPD deficiency was identified, UFT was administered at the planned dose but both the clinician and patient were informed of the risk associated with treatment and appropriate precautions were taken, such as careful monitoring of diarrhea and neutropenia.

Using a two-stage design 28 (Simon, 1989) and assuming a tumor downstaging rate of at least 25%, with alpha and beta errors of 5% and 10%, respectively, 41 patients were to be enrolled in the first stage of the study. If four or fewer responses were observed, the treatment would be considered ineffective and the trial would be stopped. If five or more responses were observed, 29 additional patients would be enrolled, for a total of 70 patients. As numerous patients were not assessable for EUS, 15 additional patients were recruited with the approval of IERB.

The primary endpoint was the tumor downstaging rate with 95% confidence interval (CI). Results are expressed as mean ± standard deviation. Quantitative variables were compared using the nonparametric Mann-Whitney test and qualitative variables using Pearson's chi-squared test or Fisher's exact test. Survival distributions were estimated using the Kaplan-Meier methodology. For safety analyses, the worst toxicity grade per patient in all chemotherapy cycles was used. All statistical analyses were conducted using Statistical Package for Social Sciences software (SPSS version 10.0, Chicago, IL, USA).

Patient characteristics at baseline are shown in Table 1. Four of the 24 known variants of the DPYD gene were identified. One patient was heterozygous for the -1590T > C SNP in the promoter region, one was heterozygous for IVS14+1G > A (G1A [DPYD*2A]), one had 2846A > T (D949V), and a fourth had 1679 T > G. A further 38 patients were heterozygous for 85 T > C. Uracil plasma levels were higher than the threshold (15 μg/L) 26 (Boisdron-Celle et al, 2007) in three of four patients with variant DPYD (IVS14+1G > A: 36 μg/L; 2846A > T: 19 μg/L; and 1679 T > G: 25 μg/L).

Sixty-three patients (74%) completed treatment as planned. Most patients (92%) received the planned dose of radiotherapy (median dose 45 Gy, range 18-46.8 Gy). Radiotherapy was postponed for at least 1 day in 66 patients (78%); dose reductions were drug related in five patients (6%). The planned UFT dose was delivered in 48 patients (mean dose 294 ± 24 mg/m²). Chemotherapy was delayed in nine patients (11%) and the dose was reduced in 15 patients (18%) as a result of adverse events, including diarrhea (n = 5), nausea/vomiting (n = 1), stroke (n = 1), dehydration (n = 3), and gastrointestinal effects (n = 1). Only four (5%) of the 85 patients did not undergo surgery: one patient died of a cerebrovascular stroke (unrelated to treatment) during the preoperative period; one patient was lost to follow-up; and the primary tumor was unresectable in two patients because of local progression. The majority of patients underwent anterior resection (AR) (n = 54; 63%); abdominoperineal resection (APR) was necessary in 27 patients (32%).

Diarrhea was the most common adverse event (60 events, 42 of which were judged to be treatment related). Most adverse events were mild to moderate in nature. Grade 3/4 adverse events, which occurred in 38 patients (45%), are shown in Table 2. Only patients who were heterozygous for variants IVS14+1G > A and 2846A > T experienced very early grade 4 neutropenia and diarrhea related to UFT. The patient who was heterozygous for variant 1679 T > G experienced grade 4 diarrhea related to treatment at week 3 and was withdrawn.

EUS was available pre- and posttreatment for 66 patients and was interpretable in 52 evaluable patients (61%). Postchemotherapy uTN staging was in agreement with double-checked pTN staging in 26 of 52 patients (50%) (Table 3). Pretreatment uTNM staging was compared with postchemoradiotherapy pTN staging (Table 4) and a significant shift towards earlier TNM stages was observed (p < 0.001). Downstaging of the primary tumor occurred in 22/52 patients (42%) and downstaging of the lymph nodes occurred in 23/52 patients (44%). In the 46 patients assessable for T and N stage, 11 patients (24%) had downstaged tumor and nodes, 11 patients (24%) had downstaged nodes only, nine patients (20%) had downstaged tumor only, and there was no difference in T or N staging in 15 patients (33%). The overall downstaging rate was 44% for primary tumors and 48% for lymph nodes. A centralized second pathologic examination performed in 78 patients confirmed the initial pTN results in 77% of cases. For nine patients, the first pathologic examination was understaged (four pT0, three pT1, two pT2, and one pT3), and for three patients it was overstaged (two pT2 and one pT4). Two patients with nonspecified T stage were classified pT3. In seven patients, malignant cells were undetectable in both the primary tumor and lymph nodes giving a pCR rate of 8%. Ten patients (12%) had some microscopic residual disease (Tmic).

Anal sphincter function was preserved in 55 patients (65%). Five of 60 patients initially deemed suitable for anterior resection (AR) had an abdominoperineal resection (APR) (8% of planned resections) and two of 23 patients whose planned surgery was APR underwent an AR (9% of planned resections). APR was performed in 60% of patients (n = 21) with a tumor in the distal third of the rectum and in 9% of patients (n = 4) with a tumor in the middle third of the rectum (within 6-8 cm of the pectineal line) due to an overestimated distance to the anal verge and to infiltration of the surgical border. AR was performed in 87% of patients (n = 41) with a tumor in the middle third of the rectum and in 40% of those whose tumor was in the distal third (n = 14) (unknown pectineal line n = 2, inextirpable n = 2). Perioperative and 30-day postoperative complications included anastomotic leakage (n = 1), perineal complications (n = 1), bleeding (n = 1), ileus (n = 1), fistula (n = 1), and death (n = 2).

The mean follow-up time was 45 months (range 1 - 84 months; one patient was lost to follow-up). OS was 92.6% at 1 year 86.1% at 2 years and 83.2% at 3 years; mean recurrence-free survival was 39 ± 24 months. Recurrence free survival was 81.9% at 1 year, 70.7% at 2 years and 66.7% at 3 years.

Two patients died within 18 days of surgery (one had a pulmonary embolism and the other had a cerebrovascular stroke). Sixteen other patients died during the study: twelwe due to progressive disease; one due to diabetic decompensation, one due to vascular cerebral stroke that occurred before surgery, and one due to acute pulmonary failure and one due to a fall.

The level of local control in the 81 patients who underwent surgery was 95%, with four patients (5%) having local recurrence and 20 (24.7%) having secondary metastases. Regardless of location, relapse generally occurred <1 year after surgery (mean: 346 ± 236 days).

Following surgery, 36 of 79 eligible patients (46%) received adjuvant chemotherapy (mean duration 118 ± 65.8 days): 21 patients (58%) received 5-FU-based chemotherapy, 10 (28%) received UFT-based chemotherapy and 4 (11) received oxaliplatin-based chemotherapy. Six patients had further chemotherapy for metastatic disease within 4 months of surgery.