Int J Colorectal Dis (2004) 19:157164 DOI 10.

1007/s00384-003-0532-x

O R I G I N A L A RT I C L E

Katsuki Ito Hiroaki Nakazato Akihiko Koike Hiroshi Takagi Shigetoyo Saji Shozo Baba Masayoshi Mai Jun-ichi Sakamoto Yasuo Ohashi

Long-term effect of 5-fluorouracil enhanced by intermittent administration of polysaccharide K after curative resection of colon cancer
A randomized controlled trial for 7-year follow-up

Accepted: 21 July 2003 Published online: 12 September 2003 Springer-Verlag 2003

S. Baba Department of Surgery 2, Hamamatsu University School of Medicine, 3600 Handa-cho, 431-3192 Hamamatsu, Japan M. Mai Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, 920-0934 Kanazawa, Japan J.-i. Sakamoto Department of Surgery, Aichi Prefectural Hospital, 18 Kuriyado, Kake-machi, 444-0011 Okazaki, Japan Y. Ohashi Department of Health Science, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113-0033 Tokyo, Japan

The authors represent the Study Group of Immunochemotherapy with PSK for Colon Cancer K. Ito () H. Takagi Department of Surgery 2, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, 466-8550 Nagoya, Japan e-mail: katsuki@med.nagoya-u.ac.jp Tel.: +81-52-7442442 Fax: +81-52-7442444 H. Nakazato Yokoyama Gastrointestinal Hospital, 3-11-20, Chiyoda, Naka-ku, 460-0012 Nagoya, Japan A. Koike Department of Surgery 1, Aichi Medical University, 21 Karimata, Yazako, Nagakute-cho, Aichi-gun, 480-1195 Aichi, Japan S. Saji Department of Surgery 2, Gifu University School of Medicine, 40 Tsukasa-machi, 500-8705 Gifu, Japan

Abstract Background and aims: The efficacy of a biological response modifier polysaccharide K in adjuvant immunochemotherapy was evaluated in primary colon cancer patients with macroscopic Dukes C

after curative resection. Patients and methods: Employing the minimization method using three factors (lymph node metastases, preoperative serum CEA level, and institution), 446 patients were allocated into groups P and C. Group P received immunochemotherapy, oral PSK (3 g per day) followed by oral 5-FU (200 mg/body per day), while group C received only intermittent chemotherapy, oral 5-FU (200 mg per day) followed by 4-week rest. Both groups received ten courses. Results: Survival for cancer death was significantly higher in group P than in group C, but there was no difference in 7-year disease-free survival or overall survival had. Conclusion: Repeated alternating administration with PSK followed by 5-FU can improve survival for cancer death. Keywords 5-Fluorouracil Polysaccharide K Colon cancer Immunochemotherapy Intermittent administration

Introduction
The death rate from malignant neoplasms has been the highest among all causes of death in Japan since it passed that of cerebrovascular disease in 1981. The total number of deaths due to cancer has reached over 280,000 annually. Lung and gastric cancer have the highest mortality rates, followed by liver and colon cancer [1]. Based on the fact that the incidence of colon cancer

is increasing (over 22,000 in deaths), in clear contrast with the decrease in gastric cancer, it appears that the order of morbidity rates by cancer site in Japan come to more closely resemble the pattern in Western countries in the near future: lung, colon, and breast cancer, respectively. To counter the rising trend in colon cancer novel therapeutic strategies for colon cancer are needed. For patients with lymph node metastasis after curative resection of colon cancer, the possibility of recurrence

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due to residual cancer cells or tumor spill during surgery must be considered. As postoperative treatment, chemotherapy and immunotherapy have been as frequently applied as radiation therapy in patients with colon cancer. More recently, immunochemotherapy, which is combined treatment with immunotherapy and chemotherapy with the intent to take advantage of the effects of each and intensify antitumor effect, is making progress worldwide. In fact host immune damage due to prolonged administration of chemotherapeutic agents can be minimized by concomitant use of immunotherapy especially with polysaccharide K (PSK) or levamisole. The protein-bound PSK extracted from mycelia of Coriolus versicolor strain CM-101, belonging to the basidiomycetes [2], is a recognized biological response modifier (BRM). It is composed of proteins and polysaccharides. Its major glycoside portion is -1,4-glucan, and it has an average molecular weight of approximately 100 kDa. PSK is a potent inducer of gene expression for several interleukins, tumor necrosis factor, and monocyte chemotactic and activating factors. The antitumor activity of PSK is thought to be based on its regulation of host immune response, and the drug is widely prescribed for oral ingestion [3]. This nonspecific immunopotentiator has the ability to inhibit the immunosuppression of cancer patients and is widely used in Japan for this purpose. PSK has been confirmed to be more effective in the treatment of gastric cancer when intermittently administered followed by alternating chemotherapy [4]. However, the immunochemotherapeutic efficacy of PSK plus antitumor agents on colorectal cancer remains unclear. With 93 cooperating institutions, all are the center hospitals of the local area, the Study Group of Colon Cancer with Immunochemotherapy was organized to evaluate the efficacy of alternating PSK followed by 5fluorouracil (5-FU) in patients with curative resection of primary colon cancer, and it conducted randomized controlled trials from 1991 to 1998. All operations were carried out by the registered surgeons to the Japan Surgical Society, and their technical abilities were approved by the society. All operations were performed along the guidelines of the operative procedures, including lymph nodes dissection, detailed in the Japanese Classification of Colorectal Carcinoma [5]. In addition to analyzing overall survival (OS), disease-free survival (DFS) and survival for cancer-related death (SCD, as an original analysis), a stratified analysis of cases of macroscopic Dukes C has been planned. The study protocol specifies that covariate analysis be considered together with the determination of subgroups after blind review. The previous review of 5-year follow-up results was presented at the 35th ASCO meeting in 1999 [6]. Since this review showed promising outcomes, extended follow-up data up to 7 years were investigated in the present study to assess the long-term effect on the clinical course of this immunochemotherapy.

Materials and methods

Organization Colon cancer patients who had undergone curative resection and had developed macroscopic lymph node metastasis were randomly assigned to receive postoperative administration of both PSK and 5-FU, or 5-FU alone as a control, at one of 93 institutions in the Chubu region of Japan between February 1991 and March 1993. The characteristics of the all patients were shown in Table 1. The protocol was approved by the institutional review board of each hospital, and informed consent was obtained from all the patients or their relatives. Eligibility The patients were assessed to be eligible when all the following conditions were met: matched: primary colon cancer with curative resection and positive macroscopic lymph node metastases (macroscopic Dukes C); under 75 years of age with PS 0 or 1; no serious complications; no preoperative treatment (radiation therapy, chemotherapy, immunotherapy) for cancer; no previous history of cancer; acceptable preoperative laboratory data (white blood cell count 3,000/mm3, platelet count 100,000/mm3, glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase 100 IU); preoperative records of serum carcinoembryonic antigen (CEA) level (below 5 ng/dl or not) and purified protein derivative (PPD) skin test reactions (smaller than 10 mm or not). A total of 441 patients were judged to be eligible according to the criteria of eligibility. Stratification and randomization procedures A chart of the randomization following registration is shown in Fig. 1. All the patients received a 48-h constant intravenous infusion of 5-FU (Kyowa Hakko Kogyo, Tokyo, Japan) at the dose of 2 1,000 mg1 m2 per 24 h [7] on postoperative days 12, 8, 9, 15, 16, 22, and 23 (if possible); that is, weekly for 34 weeks as pretreatment. The dose was supposed to be very safe and effective with a low level of adverse effects and to be associated with prolonged survival time in patients with absolutely noncurative gastric and colorectal cancer [5, 8, 9]. In the expectation of postoperative weight reduction, weight was remeasured to determine individually the amount of 5-FU after the second administration. At the point when the 5-FU infusion courses were completed the patients were stratified according to four factors: degree of histopathological lymph node metastases [5] (N0, N1, N2, N3); preoperative serum CEA level [10] (5 ng/ml, 5 ng/ml<); PPD skin test reaction (positive or negative); and institution. Patients were allocated to either the PSK group (group P, n=220) or control group (group C, n=221) according to a dynamic balancing method (modified minimization method) using three balancing factors and institution [11], based on central-registration method between 25 and 28 postoperative days. The randomized results processed in a computed system with the intention of concealment and adequate randomization, were immediately made known by telephone and/or fax to the physicians according to the treatment indication form. All operations were carried out by the registered surgeons qualified by Japan Surgical Society. The clinical characteristics of the two study groups were similar at base line (Table 1). However, there was still an imbalance in the distribution of the patients performance status (PS) between the two groups. Thus an adjusted analysis with two balancing factors (lymph node metastases, preoperative CEA level) and PS was adopted in the blinded review as a primary analysis.

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Table 1 Patient characteristics Group P (n=220) n Sex Male Female Age (years) 49 5059 6069 7075 Mean Performance status (PS) 0 1 Tumor location Cecum Ascending colon Transverse colon Descending colon Sigmoid Curability Absolute Relative PPD (balancing factor) 10 mm <10 mm CEA (balancing factor) 5 ng/ml <5 ng/ml Historical typing Well differentiated adenocarcinoma Moderately differentiated adenocarcinoma Others Depth of invasion SM MP SS SE SI Lymph node metastases (balancing factor) N0 N1 N2 N3 Dukes stage (historical) A B C % Group C (n=221) n % 0.88 (2) 123 97 28 66 85 41 60.4 186 34 29 42 18 16 115 197 23 126 94 61 159 110 96 14 4 20 133 55 8 116 63 34 7 16 100 104 55.9 44.1 12.7 30.0 38.6 18.6 121 100 35 50 107 29 60.0 199 22 18 44 29 16 114 188 33 127 94 66 155 115 95 11 4 23 140 44 10 113 66 32 10 20 93 108 54.8 45.2 0.77 (U) 15.8 22.6 48.4 13.1 0.60 (t) 0.11 (2), 0.09 (Fi) 84.5 15.5 13.2 19.1 8.2 7.3 52.3 89.5 10.5 57.3 42.7 27.7 72.3 50.0 43.6 6.4 1.8 9.1 60.5 25.0 3.6 52.7 28.6 15.5 3.2 7.3 45.5 47.3 90.0 10.0 0.27 (2) 8.1 19.9 13.1 7.2 51.6 0.26 (2) 85.1 14.9 1.00 (2) 57.5 42.5 0.70 (2) 29.9 70.1 0.79 (2) 52.0 43.0 5.0 0.37 (U) 1.8 10.4 63.3 19.9 4.5 0.73 (U) 51.1 29.9 14.5 4.5 0.91 (U) 9.0 42.1 48.9 P

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Protocol management Oral PSK (Krestin, Kureha Chemical, Tokyo, Japan) at a dose of 3 g/body per day was administered to group P from the 29th postoperative day for 4 weeks followed by 4-week oral 5-FU treatment

(200 mg/body per day) as one course. Group C received a 4-week rest and 4-week oral 5-FU chemotherapy (200 mg/body per day) as one course so that intermittent 5-FU was administered at 4week intervals. A total of ten courses were given to both groups (Fig. 2). Follow-up procedure Follow-up observation was based on the following factors: physical examination, hematological tests, blood biochemistry, check of compliance to oral treatment, assessment of postoperative complications and toxicity, initial recurrence, and outcome. Follow-up visits were requested once a month until 18 months and thereafter every 36 months up to 7 years. Abdominal computed tomography (CT) or echogram, chest radiography, and CEA/CA19-9 measurements were performed every 6 months during the follow-up period. In addition, either a barium enema or colonoscopic examination was performed every year. If recurrence was detected, the patient was not required to continue the allocated regimen but was given individually appropriate treatment. If a patient experienced toxic effects, the assigned treatment was suspended until remission. Statistical considerations In the coprimary endpoints of the study, OS, DFS, and SCD, the starting point was defined to be the day of operation. In calculating OS and DFS all deaths were treated as events in OS, whereas recurrences or deaths from all causes were treated as events in DFS. For SCD cancer death and noncancer death were defined as an event and a censored case, respectively. Noncancer death was classified as death from another disease completely or an accident unassociated with cancer-related disease. All the events of recurrence and deaths were calculated from the date of operation. Patients who were judged to be ineligible based on the information before randomization were excluded from the primary analysis, and all the other patients were included in the analysis according to the intention-to-treat principle. The required sample size was calculated to be 224 in each group, which would ensure the power of the primary log-rank test to be 80% with 0.05 type 1 error (one-sided) [12], based on the expectation that PSK would reduce the hazard rate of mortality to two thirds and on the assumption that the 7-year OS rate in the control group would be 60%. The expected number of deaths (events) was 155 in total.

Fig. 1 Registration and randomization procedure. The eligible cases were allocated into groups P or C by dynamic randomization considering the degree of histological lymph node metastases, preoperative CEA level, PPD skin test reaction, and institution Fig. 2 Modality of therapy. Patients were excluded from central registration who could not begin pretreatment of 5-FU within 7 postoperative days, or could not receive the treatment for more than 2 weeks

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All statistical analyses were carried out using SAS version 6.12. The OS, DFS, and SCD rates were calculated by the KaplanMeier method. The stratified log-rank test adjusted by the three factors was used for a comparison between groups P and C, and the similar stratified Cox regression was used for estimating risk reductions. Exclusion of PPD skin test reactions and inclusion of PS status from primary analysis were decided based on a blind review which showed the PPD skin test reaction had no influence on endpoint whereas PS had a strong effect. All P values were twosided. Additionally, the logarithmic hazard ratio was estimated for each subgroup of prognostic factors from the adjusted log-rank test, based on which the interaction test between each factor and treatment effect was performed.

cacy of PSK followed by 5-FU showed the same tendency as that seen in the PS (0) subgroup. The drug compliance levels in groups P and C were essentially the same [full compliance: 192 (87%) vs. 188 (85%); no treatment: 2 (1%) in both]. Furthermore, there was discontinuation of the therapy in 25 patients (11%) in group P and 27 (12%) in group C, ascribed to no hospital visits (group P, 11; Group C, 16), adverse events (group P, 5; group C, 4), concomitant diseases (group P, 4; group C, 2), and others (5 in both groups), suggesting no differences between the two groups (Table 2). No characteris-

Results
There were 37 cancer-related deaths (16.82%) in group P and 49 (22.17%) in group C. The major causes of twenty noncancer deaths were: myocardial infarction (n=2), cerebral infarction (n=1), cerebral hemorrhage (n=1), diabetic nephropathy (n=1), respiratory insufficiency (n=1), burn death (n=1), and suicide (n=1). The 7-year DFS of group P was statistically similar to that of group C (74.1% vs. 71.0%), and the P values calculated using the log-rank test were 0.48 (crude) and 0.10 (adjusted by the three factors), respectively (Fig. 3). The 7-year OS rates showed slight but not significant differences between groups P and C (79.6% vs. 75.6%, crude: P=0.38; adjusted: P=0.081). For 7-year SCD, however, group P was statistically higher than group C (83.4% vs. 78.5%, crude: P=0.17; adjusted: P=0.019, Fig. 4). Subgroup analysis of logarithmic hazard rates revealed that the interaction due to PS was markedly significant in SCD (Fig. 5) and was also seen in OS and DFS (P<0.047, 0.009, 0.024, respectively), suggesting that in all subgroups but those with poor PS (1) the effi-

Fig. 4 Survival for cancer death. Group P (events: 37; CDFS: 83.4%), vs. group C (events: 49; CDFS: 78.5%). Statistically significant differences were found (P=.019, log-rank test, logarithmic hazard ratio: 0.524; hazard ratio: 0.592; risk reduction: 40.8%, 95%/class I)

Fig. 3 Disease free survival. Group P (events: 60; DFS: 74.1%), vs. group C (events: 66; DFS: 71.0%). No significant differences were observed (P=0.10, log-rank test, logarithmic hazard ratio: 0.300; hazard ratio: 0.741; risk reduction: 25.9%, 95%/class I)

Fig. 5 Comparison of hazard ratio of cancer death between factors. Point estimates with 95% confidence intervals; Diff differentiated; adenoca adenocarcinoma; mod moderately. The hazard ratios were directly shifted toward minus for all factors except PS 0/1

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Table 2 Adverse effects: number and ratio (patients/year)

Group P Number Hemoglobin White blood cells Platelet Glutamic oxaloacetic transaminase Glutamic pyruvic transaminase Alkaline leukocyte phosphatase Creatinine Oral cavity disorders Loss of appetite Nausea-vomiting Diarrhea Obstipation Pain Skin disorders Others 46 84 12 44 51 47 3 1 16 13 14 37 31 6 47 Ratio 10.70 19.53 2.79 10.23 11.86 10.93 0.70 0.23 3.72 3.02 3.26 8.60 7.21 1.40 10.93

Group C Number 56 99 10 51 54 55 3 5 28 22 23 29 26 4 65 Ratio 13.08 23.13 2.34 11.92 12.62 12.85 0.70 1.17 6.54 5.14 5.37 6.78 6.07 0.93 15.19

P (2)

0.104 0.232 0.742 0.630 1.000 0.157 0.351 0.269 0.214 0.185 0.450 0.612 0.548 0.579 0.010

tic toxic effect was identified for PSK even after meticulous review of all individual medical records by the investigator in charge.

Discussion
The effectiveness of 5-FU has been reported as adjuvant chemotherapy for curatively resected colorectal carcinoma [13, 14]. In Japan there is as yet no standard chemotherapy regimen as there is in the United States. Therefore as a control group we selected the 5-FU, as a drug of adjuvant chemotherapy. Simultaneous administration of immunochemotherapy with PSK and 5-FU in patients with colon cancer has been reported, and immunochemotherapeutic advantages were confirmed in both OS and DFS [15]. The 5-year OS and DSF of adjuvant chemotherapy for curatively resected colon cancer of Dukes C patients of 5-FU vs. surgery alone were 72.8%, 55.2% vs. 61.0%, 54.5%, respectively, with statistical significance in both group [13]. Similar outcomes are suggested from studies on adjuvant immunochemotherapy using pyrimidine fluoride drugs and PSK [13, 16]. One study, however, failed to show a statistically significant difference in 6-year OS and another demonstrated significantly higher 5-year OS and DFS in all cases but failed to show significant results when the patients were stratified into subgroups of colon and rectum cancer. Furthermore, concomitant administration of 5-FU and levamisole, another BRM such as PSK in colon cancer patients with Dukes C showed significant efficacy [17] as an adjuvant therapy. However, the immunotherapeutic effect of levamisole has been controversial in recent studies [18]. Currie et al. [19] experimentally investigated the effectiveness of immunochemotherapy in various regimens including concomitant or alternating administration, and long afterward Kondo et al. [20] hypothesized that alter-

nating administration of a chemotherapeutic agent (carbazilquinone) and BRM would further optimize the antitumor effect in comparison with concomitant administration, considering the pharmacodynamic competing action between the two types of agents. Chemotherapeutic agents would suppress immune response system, while BRM would regulate or modulate the host immune response. Their results from a preclinical animal study indicated for the first time the superiority of repeating cycles of immunotherapy and chemotherapy. Adopting the modality of alternating therapy with PSK and chemotherapeutic agents including carbazilquinone, 5-DFUR (Futraful), and 5-FU as a postoperative adjuvant immunochemotherapy in patients with macroscopic curative resection of gastric and colorectal cancer, a series of studies showed significant differences between alternating administration groups (chemotherapeutic agent followed by PSK) and groups receiving intermittent chemotherapy alone, not only in individual studies but also in meta-analysis [4, 21, 22, 23]. The present 7-year follow-up study suggests that an intensified effect in terms of prolonged survival in SCD could be induced by repeated alternating administration with PSK followed by 5-FU. The regimen of oral administration would also help maintain the patients quality of life. The alternating therapy does not appear to lose its effectiveness with time, at least as far as can be determined from a comparison of the outcome between our 5year [6] and 7-year follow-up studies. That is, the 7-year SCD clearly exceeded the 5-year SCD that was adjusted by two balancing factors (lymph node metastases, preoperative CEA level) and PS (P=0.019 vs. P=0.026). Furthermore, a positive interaction was revealed between PS 0 and 1 in the analysis of logarithmic hazard ratio, suggesting that patients with PS 1 would be more sensitive to the immunomodulating effect of PSK than patients with PS 0. The orally administered dose of PSK,

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3.0 g/body per day, was the same as used conventionally [12, 24]. There may be, however, potential advantage with a greater amount of PSK in patients diagnosed as PS 1. Regarding the correlation between preoperative serum immunosuppressive acidic protein (IAP) level and the sensitivity to postoperative BRM therapy, the survival rate in the low IAP group (cutoff value 580 g/ml) was reported to be significantly higher than in the high IAP group in curatively resected gastric cancer patients [25]. Moreover, significantly longer survival outcomes were observed in the subgroup receiving immunochemotherapy than that receiving chemotherapy alone among the low IAP group, while in the high IAP group no significant tendency was observed between the immunochemotherapy and chemotherapy subgroups. Taking these observations into account, it will be necessary to investigate in future studies whether there is a correlation between PS and the degree of preoperative IAP level since the survival rates between the PS 0/1 and IAP low/high subgroups might exhibit similar tendencies. Numerous studies have investigated the immunotherapeutic actions of PSK through the pathways augmenting host immunity. Their modes of action can be defined as

radical trapping [2], modulation of cytokine production [2, 26, 27, 28] and effector cell functions [2]. Other reports have indicated that they also stimulate dendritic or Langerhans cells [29], activate killer T-cells or human natural killer cells [30, 31] and provoke secretion of cytokines [26]. However, much is unknown mechanistically about the immunotherapeutic pathways of PSK interacting with chemotherapeutic agents. The efficacy of alternating immunochemotherapy with PSK followed by other chemotherapeutic agents should be investigated to clarify the immunoactivation characteristics of PSK. Further application of the alternating therapy with other BRM may also be beneficial for prolongation of survival.

Conclusion
The repeated alternating administration with PSK followed by 5-FU would be suggested to enhance antitumor effect, resulting in a superior survival for cancer-related death compared with 5-FU alone.
Acknowledgements We thank Ms. Atsuko Tanaka for the assistance in the preparation of the manuscript.

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