© 2004 Foundation for Promotion of Cancer Research
Predictive Factors of Tumor Shrinkage and Histological Regression in Patients who Received Preoperative Radiotherapy for Rectal Cancer
Departments of 1 Surgery and 2 Radiology, Tokai University School of Medicine, Isehara, Kanagawa, Japan
For reprints and all correspondence: Toshiyuki Suzuki, Department of Surgery, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, 259-1193, Japan. E-mail: tsuzuki{at}is.icc.u-tokai.ac.jp
Received April 15, 2004; accepted October 15, 2004
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Abstract |
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 TOP Abstract INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Objective: In order to determine the reliable predictors of response to radiotherapy for rectal cancer, we assessed apoptosis, p53 and p21 in biopsy specimens collected before treatment, and investigated the relationships to the histological effect of irradiation and the degree of tumor shrinkage.
Methods: Ninety-three patients with advanced rectal adenocarcinoma were treated with preoperative irradiation of 20 Gy in 10 fractions in combination with intraoperative electron beam irradiation of 15 Gy. We evaluated tumor grade, pathological tumor regression, biological markers associated with apoptosis and proliferation [apoptotic index (AI), p53, p21 and Ki67], and the degree of tumor shrinkage.
Results: Considering positivity of p21 and of apoptosis and negativity of p53 as favorable factors in tumor shrinkage, we compared the degree of shrinkage among the patients using the number of favorable factors as the score. The degree of shrinkage was 41.5 ± 8.5% in patients with three favorable factors, 31.4 ± 9.7% in patients with two favorable factors and 26.5 ± 11.2% in patients with one favorable factor. However, there was no significant difference in the proportion of marked regression according to the number of favorable factors.
Conclusions: Histological examination of apoptosis, p21 and p53 in biopsy specimens and scoring were considered to be useful predictive methods for assessing the efficacy of radiotherapy for rectal cancer.
Key Words: rectal cancer • p53 • p21 • apoptosis • radiation sensitivity
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INTRODUCTION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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The rate of local recurrence has been reported to be from 19 to 26% in patients with advanced rectal cancer (1,2). A meta-analysis of 14 randomized control trials showed that preoperative radiotherapy significantly decreases local recurrence (3). Short-term high-dose preoperative radiotherapy significantly improved survival rate, whereas it increased morbidity (4,5). On the other hand, total mesorectal excision (TME) alone has been reported to decrease the rate of local recurrence to
10% (6,7). TME combined with preoperative radiotherapy has been reported to reduce the 2-year local recurrence rate to 2.4% compared with 8.2% for TME alone (8). However, the combination therapy increased the complication rate (9). In order to prevent (the) local recurrence without increasing complications, we conducted preoperative radiotherapy of 20 Gy in 10 fractions in combination with intraoperative radiotherapy (IORT) based on a new concept that 15 Gy of electron beam is delivered equally to the entire surface of the pelvic wall following removal of the rectum. We reported that this method decreased the rate of local recurrence to 3% and achieved a significantly good survival rate in patients with stage II or III rectal cancer (10).
As for the correlation between the effect of preoperative irradiation and prognosis, disease-free survival is reported to be significantly better in patients in whom histological downstaging or histological regression was observed after irradiation (11,12). Preoperative irradiation enhances the possibility of a sphincter-preserving operation in patients with marked histological effects (12). Therefore, it will be clinically significant to identify indicators that predict histological regression and the degree of tumor shrinkage before preoperative radiotherapy. Willett et al. (13,14) reported that patients with small size tumor and elevated proliferating cell nuclear antigen (PCNA) and mitosis levels showed a high rate of local downstaging. However, currently there are no reliable predictors of response to radiotherapy that can easily be evaluated before starting irradiation. Since the effect of radiation is mediated by apoptosis induced by DNA damage (15), p53 and p21 of apoptosis-related genes may be predictive factors of radiation sensitivity (16). In the present study, we immunohistologically assessed p53, p21, apoptosis, and Ki67 in biopsy specimens taken before irradiation and resected specimens, and investigated their relationships with histological effects and degree of tumor shrinkage to evaluate their significance as predictive markers of response to radiotherapy.
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SUBJECTS AND METHODS |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Between 1991 and 2001, 93 patients with histologically proven adenocarcinoma of the middle or lower third of the rectum were treated with surgery in combination with preoperative radiotherapy. Preoperative diagnosis was T3/T4, Nx, M0, according to the TNM classification (UICC, 1997).
The initial evaluation included digital examination of the rectum, chest X-ray, colonoscopy, double contrast barium enema, and computed tomography of the abdomen and pelvis. Endorectal ultrasound has also been used since 1994. There were 72 males and 21 females; mean age was 59.4 ± 10.5 years (range 34–89); tumor location was the middle third of the rectum in 29 patients and the lower third of the rectum in 64 patients.
Tumor location was determined according to the criteria of the Japanese Society for Cancer of the Colon and Rectum (17). The upper third of the rectum is defined as the area between the promontory and the inferior margin of the second sacral vertebra on the lateral view of a barium enema examination. The remaining rectum is divided into the middle and lower third of the rectum by the peritoneal reflection. The level of the peritoneal reflection corresponds to the level of the middle Houston valve on barium enema images.
Preoperative radiotherapy was performed with 18 MV X-ray by a linear accelerator (Clinac 2100C, Varion Med System, Inc. Palo Alto, CA) using the two-field technique (anterior–posterior and posterior–anterior fields). Irradiation was carried out once (2 Gy) daily with a total dose of 20 Gy. Surgery was done 2 weeks (range 9–18 days) after completion of the irradiation, and 15 Gy of electron beam was delivered during surgery. Double contrast barium enema examinations were performed before irradiation and immediately before surgery to calculate the degree of tumor shrinkage by measuring the tumor along the major axis on the lateral views. Each measurement was corrected by the diameter of the first sacrum (Fig. 1). A rate of tumor shrinkage of 
30% was evaluated as response to treatment.
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The biopsy specimens before irradiation were collected using a colonoscopy. The biopsy specimens and surgically resected specimens were fixed with formalin, embedded in paraffin, and sectioned into 6 µm slices. Apoptotic cells were stained using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The number of nuclei of positive tumor cells per 1000 tumor cells was counted to calculate the apoptotic index (AI). An AI
3% was considered as positive. Expression of p53, p21 and Ki67 was evaluated by immunohistological staining (indirect method) using DO7 (Novocastra Laboratories, Newcastle upon Tyne, UK), Cip1/WAF1 70 (BD Biosciences, CA) and MIB-1 (DAKO, Glostrup, Denmark) as primary antibodies, respectively. The percentage of positive cells per 1000 tumor cells was regarded as a labeling index, and p53 35%, p21 10% and Ki67 60% were considered as positive. Histological regression was diagnosed according to three different criteria (17–19). In the Japanese Classification of Colorectal Carcinoma (17), grade 2 (moderate change: necrosis or disappearance of the tumor is present in more than two-thirds of the whole lesion, but viable tumor cells still remain) or more regression (grade 3) was considered as marked regression. Grade 3 (there were several residual cells, but the fibrosis predominates) or more regression (grade 1 and 2) in Tumor Regression Grade (TRG) (18), and grade 2 (isolated microscopic foci of tumor less than three) or more regression (grade 3 and 4) in the method of Rich et al. (19) was considered as marked regression, respectively. Histological regression was assessed on the central section of each tumor stained with hematoxylin and eosin.
Statistical analysis was performed by Student t-test Mann-Whitney U-test, 
2 test and Peason's correlation coefficient at a 5% significance level.
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RESULTS |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Immunohistological evaluation could be carried out in biopsy specimens of all 93 patients whereas in surgically resected specimens, it was performed for 89 patients because complete or almost complete regression was obtained in four patients. The degree of shrinkage on average was 26.7 ± 12.4% (mean ± SD). The degree of shrinkage was not different between the middle third and lower third of the rectum. There was also no difference in response rate between these parts of the rectum. Although only a few (four) patients had poorly differentiated adenocarcinoma, the degree of shrinkage in poorly differentiated adenocarcinoma was significantly larger than that of moderately differentiated adenocarcinoma (P = 0.03). The response rate was also higher in poorly differentiated adenocarcinoma (Table 1).
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Although the tumor size of the excised specimens was smaller by 4% compared with the measurement values on the double contrast barium enema, each value was well correlated, with a Pearson's correlation coefficient of 0.88 (P < 0.0001) (Table 2).
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The degree of shrinkage was significantly greater in p53-negative than p53-positive cases (P = 0.002). The same was true for p21-positive cases compared with negative cases (P = 0.0006), and for apoptosis-positive cases compared with negative cases (P = 0.0001).
There were significantly more responses in p53-negative cases compared with positive cases (P = 0.02). Similarly, there were more responses in p21-positive cases compared with negative cases (P = 0.05), and in apoptosis-positive cases compared with negative cases (P < 0.0001). However, no correlation was noted between Ki67 values and response rates (Table 3).
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In the assessment by the Japanese Classification of Colorectal Carcinoma (17), the frequency of marked histological regression in patients with positive p21 and with positive apoptosis was 61.5 and 55.6%, respectively, being significantly higher than that in patients with negative p21 and with negative apoptosis (P = 0.01 and P = 0.005). In the assessment by the TRG (18), the frequency of marked regression in patients with positive p21 and with positive apoptosis was 69.2 and 62.2%, respectively, being significantly higher than that in patients with negative p21 and with negative apoptosis (P = 0.04 and P = 0.05). In the assessment by the method of Rich et al. (19), the frequency of marked regression in patients with positive apoptosis was 77.8% and was significantly higher than that in patients with negative apoptosis (P = 0.01). Apoptosis was considered to be related to histological effects in the three assessments above. However, p21 expression was related to histological effects in only two assessments by the Japanese Classification of Colorectal Carcinoma and TRG. There was no relationship between p53 or Ki67 expression and histological effect in all of the above three assessments (Table 4).
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Marked hitological regression was significantly related to tumor shrinkage and response rate by all of the three criteria (P
0.03) (Table 5).
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The positivity of Ki67 expression was 64.0% before irradiation and 15.7% after irradiation, and thus decreased significantly after irradiation (P < 0.0001). The positivity of p21 also decreased significantly after irradiation (P < 0.0001). The positivity of apoptosis induction was 46.1% before irradiation and 85.4% after irradiation, and thus increased significantly after irradiation (P < 0.0001). There was no significant change in p53 positivity after irradiation (Table 6).
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The relationship between p53 expression and p21 expression in biopsy specimens before irradiation was not clear in our immunological evaluation (Table 7). Since positivity of p21 and of apoptosis and negativity of p53 in the biopsy specimens before irradiation seemed to be favorable factors for shrinkage, we compared the degree of shrinkage, response rate and histological regression among the patients using the number of favorable factors as the score. The degree of shrinkage was 41.5 ± 8.5% in patients with three favorable factors, 31.4 ± 9.7% in patients with two favorable factors and 26.5 ± 11.2% in patients with one favorable factor (three versus two, P = 0.007; three versus one, P = 0.0003) (Table 8).
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However, there was no significant difference in the proportion of marked regression among three groups classified according to the number of favorable factors.
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DISCUSSION |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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Preoperative radiotherapy and chemoradiotherapy for rectal cancer have been reported to reduce the incidence of local recurrence and to increase sphincter-preserving surgery (3,12). We have performed combined treatment of preoperative radiotherapy, radical surgery and IORT for resectable rectal cancer (20). We developed a new concept of creating a tumor-free margin by IORT to suppress local recurrence (20,21). Our target sites of IORT were the resected surface of the pelvic wall and the tissues 1–2 cm beneath it. Therefore, the electron beams irradiated as uniformly as possible the entire dissected surface of the pelvic cavity after resection of the rectal cancer. We used a dose of 20 Gy in 10 fractions for preoperative radiation. The biological effectiveness of single-dose IORT is considered to be equivalent to two or three times the dose of fractionated electron beam radiotherapy (EBRT) (22). Thus if 15 Gy of IORT is added to a preoperative dose of 20 Gy delivered as EBRT, this should equal at least 50 Gy. We had reported previously that this therapy modality reduced the local recurrence at 3% with perioperative morbidity at 34%, which was the same as that in patients who underwent surgery alone (20). If tumor response to preoperative radiothearpy could be predicted before the treatment, it would be feasible to select a suitable treatment plan for each patient.
Berger et al. (11) reported that preoperative radiotherapy was more effective in patients with well differentiated adenocarcinoma than in patients with moderately differentiated adenocarcinoma. Although this was also found in the present study, the difference was marginal (P = 0.06) (Table 1).
As for histological effect as prognostic factor, Willett et al. (13,14) reported that PCNA and Ki67 levels in tumors were reduced after irradiation, that PCNA and Ki67 levels before irradiation in patients with remarkable tumor regression were higher than those in patients without remarkable tumor regression, and that patients with elevated PCNA and mitosis levels in their resected specimens had a better prognosis. In our study, Ki67 expression, which is a proliferation index similar to PCNA, was significantly reduced after irradiation (P < 0.0001), but a relationship between Ki67 expression in the biopsy specimens before irradiation and the degree of shrinkage was not found (Tables 3 and 6).
Generally speaking, detection of wild-type p53 protein by immunohistological methods is difficult because it occurs in very small amounts and has a short half-life. However, the half-life of mutant p53 protein is longer and thus mutant p53 in tumor tissue can be easily detected by immunohistochemical staining. Therefore, most of the p53 protein detected by immunohistochemical staining is considered as mutant p53 protein (23).
The effects of anticancer drugs and radiation are considered to be produced by apoptosis induction via activation of p21 caused by wild-type p53. Mutant p53 lacks this function (16,24). p53 and p21, cancer suppressor genes, are closely related to each other in the apoptosis pathway. However, the relationship between p53 expression and p21 expression was not clear in our immunological evaluation (Table 7). Thus, it is likely that a p53-independent apoptosis pathway might be induced by radiation in patients with positive p53 who showed marked regression of their tumor due to radiation (25).
Fu et al. (26) reported that preoperative irradiation of 50 Gy produced a marked histological effect in patients with negative p53 and with positive p21 in their preoperative tissue specimens. In the present study, patients with negative p53 in their biopsy specimens collected before radiotherapy showed a high rate of tumor shrinkage compared with patients with positive p53 (P = 0.002) (Table 3), but no significant difference in histological effect was found (Table 4). Francois et al. (27) reported that the percentage of patients showing downstaging was 26% in patients who underwent surgery 6–8 weeks after irradiation, whereas this was lower (10%) in patients who underwent surgery 2 weeks later. Achievement of histological regression seems to require a long interval between the completion of irradiation and surgery. The inconsistency between the present results and those of Fu et al. (26) might be caused by a shorter interval in our series (average 14 days) compared with the interval in Fu et al.'s study (average 33 days).
In Western countries, the TRG (18), which was originally produced for grading of esophageal carcinoma, and Rich et al.'s classification (19), based on the distribution of the remaining cancer cells, were used as evaluation of the histological effect after irradiation in patients with rectal cancer. Table 4 shows the relationship between Ki67, p53, p21, apoptosis and histological effects according to three different methods The relationship between radiation effect and apoptosis was found using all classifications, but the relationship between radiation effect and p21 was not found in Rich et al.'s classification (Table 4). This may be attributed to patients in whom the irradiation effect was evident but a few cancer cells still remained in the deep area. They are classified as a class of lower irradiation effect in Rich et al.'s classification. Since criteria for evaluating the radiation effect in rectal cancer were thought to be favorable to assess the whole tumor, we considered the Japanese Classification of Colorectal Carcinoma or TRG to be more suitable. In all of the three classifications, marked regression was significantly related to tumor shrinkage and response rate (P 0.03) (Table 5).
In the present study, we evaluated the degree of tumor shrinkage as an indicator of radiological effect in addition to histological regression. Because the tumor size of the excised specimens and the measurement values on the double contrast barium enema were well correlated (P < 0.0001) (Table 2), the degree of tumor shrinkage calculated from the results of the double contrast barium enema is considered to be reliable.
The effects of radiotherapy on tumors are divided into two: one is the effect on a tumor growing through the bowel wall, into the mesorectum or into the neighboring structures; and the other is the effect on the longitudinal dimension of tumors. The shrinkage of a tumor assessed on the lateral view of double contrast barium enema examination can be assessed preoperatively, and is valuable as an evaluation index of the radiation effect.
CONCLUSIONS
Preoperative irradiation for advanced rectal cancer produced a significantly large degree of shrinkage and a significantly high frequency of histological regression in patients with negative p53, with positive p21, and with positive apoptosis in their biopsy specimens collected before irradiation. Although a prospective study will be needed to prove the results, a scoring system based on histological staining for apoptosis, p21 and p53 in preoperative biopsy specimens will be useful as predictive indicators for assessing the efficacy of radiotherapy in rectal cancer.
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References |
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TOPAbstractINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONReferences |
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