INTRODUCTION. Rectal cancer/hypoxia-inducible factor 1α/Preoperative chemoradiotherapy/hyperthermia/recurrence-free

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1 J. Radiat. Res., 52, (2011) Expression of Hypoxia-inducible Factor 1α Predicts Clinical Outcome after Preoperative Hyperthermo-chemoradiotherapy for Locally Advanced Rectal Cancer Mariko SHIOYA 1, Takeo TAKAHASHI 1 *, Hitoshi ISHIKAWA 1, Hideyuki SAKURAI 2, Takeshi EBARA 1, Yoshiyuki SUZUKI 1, Jun-ichi SAITOH 1, Tatsuya OHNO 1, Takayuki ASAO 3, Hiroyuki KUWANO 3 and Takashi NAKANO 1 Rectal cancer/hypoxia-inducible factor 1α/Preoperative chemoradiotherapy/hyperthermia/recurrence-free survival. Hypoxia-inducible factor 1α (HIF-1α) is an intrinsic marker of tumor hypoxia. It has been considered that hypoxic conditions reduce radiosensitivity, but the role of HIF-1α in patients treated with preoperative therapy for rectal cancer is still unclear. The aim of this study was to evaluate the predictive value of tumor response to preoperative hyperthermo-chemoradiotherapy (HCRT) and the prognostic significance of HIF-1α expression in patients with locally advanced rectal cancer. Between 2003 and 2006, 50 patients with histologically proven rectal adenocarcinoma who underwent HCRT followed by surgery were investigated. HIF-1α expression was immunohistochemically evaluated using pre-treatment biopsies. The total radiation dose was Gy and chemotherapy consisted of 5-FU and LV administered by continuous infusion on Day 1 5, Day 15 19, and Day during radiotherapy. Hyperthermia treatment was performed for once a week for 2 5 sessions. The surgical operation was performed 8 weeks after HCRT and each resected specimen was graded by histological criteria of the Japanese Classification of Colorectal Carcinoma. The effects of HIF-1α on clinical outcomes were analyzed by univariate and multivariate analysis. Positive HIF-1α expression was recognized in 42.0% of samples (21/50). Resected specimens that showed pathological grades 1, 2, and 3 numbered 17, 24, and 9 cases, respectively. There were no significant differences between the HIF-1α-positive group and HIF-1α-negative group for pathological grading and pcr. Overall survival (OS) rate at 3 years in the HIF-1α-negative group was 85.2%, which was significantly better than the 60.6% in the HIF-1α-positive group. Recurrence-free survival (RFS) rate at 3 years in the HIF-1α-negative group was 82.8%, being significantly better than 47.6% in the HIF-1αpositive group. In addition, elevated HIF-1α expression was significantly correlated with recurrence-free survival and metastasis-free survival rate in multivariate analysis. HIF-1α expression might be predictive of recurrence-free survival and metastasis-free survival rate for rectal cancer patients treated with HCRT. INTRODUCTION *Corresponding author: Phone: , Fax: , taketaka@saitama-med.ac.jp 1 Department of Radiation Oncology, Gunma University Graduate School of Medicine, , Showa-machi, Maebashi, Gunma , Japan; 2 Department of Radiation Oncology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1, Tennoudai, Tsukuba, Ibaraki , Japan; 3 Department of General Surgical Science, Gunma University Graduate School of Medicine, , Showa-machi, Maebashi, Gunma , Japan. doi: /jrr Preoperative radiotherapy, administered alone or in combination with chemotherapy, for locally advanced rectal cancer (LARC) has been established as a neoadjuvant treatment in Europe and the United States. 1 6) Preoperative chemoradiotherapy (CRT) can lead to tumor downstaging, which is correlated with resectability, improved survival, decreased local recurrence, and a higher rate of sphincter-preserving surgeries. 7 9) It is reported that the response to preoperative CRT significantly improved survival of locally advanced rectal cancer. 9) Because intensifying neoadjuvant therapy seems to be important for improving clinical results, we used local hyperthermia treatment combined with preoperative CRT. Hyperthermia has a cytocidal effect on cells in hypoxic environments that are specifically found in tumor tissue. In addition, hyperthermia increases the radiosensitivity, especially for hypoxic and nutrient-deprived cells in an acidic environment. 10)

2 822 M. Shioya et al. Hypoxia is an important environmental regulator of tumor angiogenesis and growth and modulates radiation sensitivity. In experimental studies, cancer cells in a hypoxic condition are times more radioresistant than those in an oxic condition. 11) Tumor hypoxia is a well-known factor linked to poor response to radiotherapy. Many of the adaptations to hypoxia in cancer cells are mediated by the activation of specific genes through hypoxia-inducible factors (HIFs); the transcription factor HIF-1α is a key factor in the adaptations and is an oxygen-regulated component that determines HIF activity. 12) HIF-1α targets regulate several important processes including erythropoiesis, metabolism, angiogenesis, invasion, proliferation, and cell survival. 13) There are several reports that tumors expressing HIF-1α showed poorer prognosis after radiation therapy than HIF-1α-negative tumors in head and neck cancer, cervical cancer, esophageal cancer and others ) However, few studies have reported the prognostic significance of HIF-1α expression in radiation therapy for patients with rectal adenocarcinoma. Moreover, whether hyperthermia can overcome radiation resistance induced by hypoxia or not in a clinical setting is still unclear. The aim of the present study is to determine whether the expression of HIF-1α in pre-treatment rectal cancer biopsy specimens predictive of tumor response to preoperative hyperthermo-chemoradiotherapy (HCRT) and the relationship between HIF-1α expression and survival. MATERIALS AND METHODS Patients Between 2003 and 2006, consecutive 50 patients with histologically proven rectal adenocarcinoma who underwent HCRT followed by surgery were investigated. For diagnostic workup, all patients underwent staging for regional and distant metastases with computed tomography (CT) of the abdomen and thorax. The pathology of all tumors was adenocarcinoma. T stage was determined by CT and magnetic resonance imaging (MRI), especially T2 weighted image. The extent and location of the tumor were classified according to TNM classification of malignant tumors. Patient characteristics are summarized in Table 1. The median age of the patients was 59 years old (range years old) at the beginning of the treatment. The median follow-up was 31 months (range 3 59 months) for all patients. Treatments All patients in this study underwent preoperative chemoradiotherapy combined with hyperthermia treatment. External beam radiotherapy was delivered by 10 MV X-ray through an anteroposterior or three- or four-field box technique. The clinical target volume (CTV) encompassed the primary tumor and entire mesorectal tissue. The total radia- Table 1. Correlation between hypoxia-inducible factor 1α expression and various clinical variables. Number of patients All (n = 50) Positive (n = 21) Negative (n = 29) p value Age (years) median (range) 59 (37 77) 60 (38 72) 57 (37 77) 0.90 Gender male (%) 38 (76) 14 (67) 24 (83) 0.33 female (%) 12 (24) 7 (33) 5 (17) T factor T2 (%) 6 (12) 2 (9) 4 (14) 0.10 T3 (%) 38 (76) 14 (67) 24 (83) T4 (%) 6 (12) 5 (24) 1 (3) N factor positive (%) 35 (70) 17 (81) 18 (62) 0.26 negative (%) 15 (30) 4 (19) 11 (38) Radiation dose (Gy) average (range) 48.4 (40 50) 49.0 (40 50) 47.9 (40 50) 0.27 CT course average (range) 2.5 (2 3) 2.6 (2 3) 2.4 (2 3) 0.19 Treatment time of HT (mimutes) average (range) 264 ( ) 263 ( ) 264 ( ) 0.94 (CT: chemotherapy, HT: hyperthermia)

3 HIF-1α and HCRT for Rectal Cancer 823 tion dose was Gy, with daily fractions of 2 Gy. Chemotherapy consisted of 5-FU (250 mg/m 2 per day) and leucovorin (LV) (25 mg/m 2 per day) administered by continuous infusion on Day 1 5, Day 15 19, and Day during radiotherapy. Hyperthermia was performed for once a week for 2 5 sessions (median 5 sessions) with 8 MHz radiofrequency capacitive heating equipment (Thermotron-RF 8, Yamamoto Vinita Co. Ltd., Japan). Surgery, which was mainly total mesorectal excision, was performed 8 weeks after the completion of HCRT. Assessment of HIF-1α expression Immunohistochemical staining of the paraffin-embedded specimens for HIF-1α was performed by the streptavidinbiotin complex method. Three-μm-thick sections were deparaffinized with xylene then rehydrated. After rehydration through a graded ethanol series, the tissue sections were autoclaved in target retrieval solution (DAKO, Glostrup, Denmark) at 93 C for 75 min, cooled to 30 C, and then incubated with fresh 0.3% hydrogen peroxide in methanol for 30 min at room temperature. After incubation with normal donkey serum for HIF-1α slides for 30 min, the normal donkey serum on the tissue sections was removed by blotting. The sections were then incubated with the primary anti- HIF-1α monoclonal antibody (Neomarkers, Fremont, CA) at a dilution of 1:20000 in phosphate-buffered saline (PBS) containing 10% donkey serum albumin at 4 C overnight, washed in PBS, and incubated with secondary antibody for 30 min at room temperature. Immunohistochemistry was performed with universal LSAB+ Kit/HRP (DAKO, Glostrup, Denmark). After washing PBS, the peroxidase (0.0055%) reaction was run for 3 min with 3.3 -diaminobenzidine (DAB). The sections were lightly counterstained with hematoxylin. Negative controls were prepared by substituting PBS for each primary antibody, and no detectable staining was confirmed. Uterine cervical cancer tissue sections with strong nuclear HIF-1α expression were used as positive controls. Nuclear expression of HIF-1α was determined by assessing semiquantitatively the percentage of decorated tumor cells. The samples were divided into two groups of negative vs. positive reactivity. Cases with over 40% positive cells were rated as positive. All slides were examined and scored independently by two investigators. Histopathological response for HCRT Each resected specimen was examined in terms of the histological changes after HCRT according to the histological criteria of the Japanese Classification of Colorectal Carcinoma. 18) The grades are judged according to the amounts of necrosis, degeneration, and/or lytic change of the tumor as estimated proportions of the total amount of the lesion. Grading of histopathological response was performed by a pathologist blinded to the immunohistochemistry results. Statistical analysis All data were analyzed using either Fisher's exact t test or Yates continuity-corrected chi-square test. Overall survival, recurrence-free survival, and metastasis-free survival were calculated from the first day of HCRT. Overall survival was defined as the time from random assignment to death as a result of all cases. Recurrence-free survival and metastasisfree survival were defined as the time from random assignment to recurrence and metastasis, respectively. Survival rates were plotted using the Kaplan-Meier actuarial method and the log-rank test was used to determine significance. The parameters were also analyzed by multivariate analysis using Cox s proportional hazard model. Differences were considered significant if the p-values were below RESULTS HIF-1α expression and clinical parameters Figure 1 shows immunohistopathological features. HIF- 1α immunostaining was detected in cancer cells in tumor tissues and localized predominantly in their nucleus (Fig. 1(a)). Fig. 1(b) shows negative staining of HIF-1α. All positive cells revealed strong nuclear HIF-1α immunostaining. Pos- Fig. 1. Immunohistopathological features of hypoxia-inducible factor 1α. (a) A representative specimen of strong expression of hypoxia-inducible factor 1α in rectal adenocarcinoma: 400. (b) A representative specimen of negative expression of hypoxia-inducible factor 1α in rectal adenocarcinoma: 400.

4 824 M. Shioya et al. itive HIF-1α expression was recognized in 42.0% of patients (21/50). HIF-1α expression was not correlated with age, gender, T factor, or N factor (Table 1). In addition, there was no significant difference between patients positive and negative for HIF-1α expression in terms of radiation dose, chemotherapy course, or treatment time of hyperthermia (Table 1). Correlation between HIF-1α expression and pathological evaluation Resected specimens that showed pathological grades 1, 2, and 3 numbered 17, 24, and 9 cases, respectively (Table 2). Pathological complete response (pcr) and non-pcr were 18% and 82%, respectively. There were no significant differences between the HIF-1α-positive group and the HIF- 1α-negative group in terms of pathological grade or pcr. Correlation between HIF-1α and survival Figure 2 shows overall survival (OS) rates according to HIF-1α expression. The overall survival rate at 3 years in the HIF-1α-negative group was 85.2%, which was significantly better than 60.6% in the HIF-1α-positive group. Figure 3 shows recurrence-free survival (RFS) rate according to HIF- 1α expression. The patterns of failure after treatment were 1 locoregional recurrence, 2 locoregional recurrence plus distant metastasis, and 13 distant metastases. The recurrence-free survival rate at 3 years in the HIF-1α-negative group was 82.8%, being significantly better than 47.6% in Table 2. Correlation between hypoxia-inducible factor 1α expression and pathological evaluation. Number of patients (%) All (n = 50) Positive (n = 21) Negative (n = 29) p value T factor down stage yes 28 (56) 11 (52) 17 (59) no 22 (44) 10 (48) 12 (41) 0.66 ccr yes 12 (24) 4 (19) 8 (28) no 38 (76) 17 (81) 21 (72) 0.72 pathological grade grade 1 17 (34) 6 (29) 11 (38) grade 2 24 (48) 13 (62) 11 (38) 0.20 grade 3 9 (18) 2 (9) 7 (24) pcr yes 9 (18) 2 (10) 7 (23) no 41 (82) 19 (90) 22 (77) 0.34 (ccr: clinical complete response, pcr: pathological complete response) Fig. 2. Overall survival curves of patients with rectal cancer according to hypoxia-inducible factor 1α expression. There was a statistically significant difference in the overall survival. Fig. 3. Recurrence-free survival curves of patients with rectal cancer according to hypoxia-inducible factor 1α expression. There was a statistically significant difference in the recurrence-free survival.

5 HIF-1α and HCRT for Rectal Cancer 825 the HIF-1α-positive group. In univariate analysis, radiation dose, treatment time of hyperthermia, and HIF-1α expression were found to be correlated significantly with recurrence-free survival (Table 3(a)). On the other hand, radiation dose and treatment time of hyperthermia significantly correlated with metastasis-free survival rate (Table 3(b)). As for significant factors on univariate analysis, multivariate analysis was performed. HIF-1α expression was a significant prognostic factor for recurrence-free survival rate (Table 4(a)) and metastasis-free survival rate (Table 4(b)). Hyperthermia was a significant prognostic factor for metastasis-free survival rate (Table 4(b)). Table 3(a). Univariate analysis of prognostic factors for recurrence-free survival rate after hyperthermo-chemoradiotherapy. Factors 3-year rates (%) p value Age (years) 59 (n = 25) 72.0 ± (n = 25) 64.6 ± 18.8 Gender Male (n = 38) 68.4 ± Female (n = 12) 66.7 ± 26.7 Stage I II (n = 15) 73.3 ± III (n = 35) 65.7 ± 15.7 Radiation dose (Gy) 49 (n = 8) 37.5 ± (n = 42) 73.8 ± 13.3 CT course 2 (n = 26) 57.7 ± (n = 24) 79.2 ± 16.2 Treatment time of HT (minutes) 259 (n = 16) 50.0 ± (n = 34) 76.5 ± 14.2 HIF-1α expression Positive (n = 21) 47.6 ± Negative (n = 29) 82.8 ± 13.7 (CT: chemotherapy, HT: hyperthermia, HIF-1α: hypoxiainducible Table 3(b). Univariate analysis of prognostic factors for metastasis-free survival rate after hyperthermo-chemoradiotherapy. Factors 3-year rates (%) p value Age (years) 59 (n = 25) 72.0 ± (n = 25) 71.0 ± 18.1 Gender Male (n = 38) 73.0 ± Female (n = 12) 66.7 ± 26.7 Stage I II (n = 15) 73.3 ± III (n = 35) 70.7 ± 15.3 Radiation dose (Gy) 49 (n = 8) 37.5 ± (n = 42) 78.0 ± 12.7 CT course 2 (n = 26) 61.3 ± (n = 24) 82.6 ± 15.5 Treatment time of HT (minutes) 259 (n = 16) 50.0 ± (n = 34) 81.7 ± 13.2 HIF-1α expression Positive (n = 21) 54.7 ± Negative (n = 29) 82.8 ± 13.7 (CT: chemotherapy, HT: hyperthermia, HIF-1α: hypoxiainducible Table 4(a). Multivariate analysis of prognostic factors for recurrence-free survival rate after hyperthermo-chemoradiotherapy. Recurrence-free survival rate Factors Hazard ratio 95%C.I. p value Radiation dose (Gy) CT course Treatment time of HT (minutes) HIF-1α expression (CT: chemotherapy, HT: hyperthermia, HIF-1α: hypoxia-inducible Table 4(b). Multivariate analysis of prognostic factors for metastasis-free survival rate after hyperthermo-chemoradiotherapy. Metastasis-free survival rate Factors Hazard ratio 95%C.I. p value Radiation dose (Gy) CT course Treatment time of HT (minutes) HIF-1α expression (CT: chemotherapy, HT: hyperthermia, HIF-1α: hypoxia-inducible

6 826 M. Shioya et al. DISCUSSION Rectal cancer in the lower part of the rectum has a high rate of local recurrence with poor prognosis, and the quality of life after radical surgical procedures, such as lateral node dissection, which have a limited survival rate, is severely deteriorated. 19) Surgery combined with radiotherapy is a standard treatment for patients with stage II/III rectal cancer in the United States and Europe. Recently, neoadjuvant CRT has been increasingly used for LARC and approximately 45% of LARC patients respond to neoadjuvant CRT in terms of downstaging by at least one T stage. 20) However, there is no useful molecular biomarker to predict the outcome after neoadjuvant CRT. It is well known that hypoxia is associated with reduction in radiosensitivity and causes recurrence after radiotherapy ) In addition, some analyses reported that poor prognosis resulting from hypoxia was associated not only with local failure but also with distant metastases ) HIF-1α is regarded as a hypoxic marker and as playing an important role in hypoxia. It was associated with poor prognosis and response to radiotherapy in esophageal cancer, head and neck cancer, and uterine cervical cancer. 14,15,28) However, the role of HIF-1α as a prognostic marker in patients with rectal cancer has not been comprehensively investigated. In the present study, the patients with HIF-1α expression tended to have a poor prognosis after HCRT for rectal cancer. In particular, our results suggest that HIF-1α expression is a marker of not only recurrence-free survival but also metastasis-free survival. Dellas et al. reported that elevated HIF-1α expression was associated with local tumor progression and increased rate of metastasis in advanced cervical cancer prior to radiotherapy. 16) Lu et al. demonstrated that high HIF-1α expression of rectal adenocarcinoma patients was significantly correlated with shorter overall survival and poorer local control. 29) Bachtiary et al. described that strong expression of HIF-1α was also an independent prognostic factor for shorter progression-free survival in patients undergoing radiotherapy for uterine cervical cancer. 15) Ishikawa et al. reported that recurrence-free survival and metastasis-free survival in HIF-1α-positive patients were also significantly poorer than in HIF-1α-negative patients for stage IIIB cervical carcinoma. 17) These results suggested that HIF-1α expression was correlated with not only local recurrence but also distant metastasis for a variety of cancers. The oxygenation status of a tumor, tumor hypoxia grade, is known to be an independent prognostic factor for local control in uterine cervical cancer. 16,30) On the other hand, hyperthermia improves the efficacy of radiotherapy in hypoxic cells of rectal cancer. 31) Mild hyperthermia is considered to dilate tumor blood vessels, increase tumor oxygenation, and enhance tumor radiosensitivity. 32) Sun et al. reported that tumor hypoxic fraction was decreased during and after mild temperature hyperthermia in a xenograft model. 33) In addition, mild hyperthermia at 42 C or less enhanced the therapeutic effect of anticancer drugs. 34) In rectal cancers, hyperthermia also had an additional effect when added to radiotherapy in the treatment of advanced rectal cancer. 31) You et al. showed that the pathological CR rate in a thermoradiotherapy group was significantly higher than that in a radiotherapy group for rectal cancer preoperatively. 35) In this study, the treatment time of hyperthermia was correlated with recurrence-free survival and metastasis-free survival. Our results suggested that hyperthermia had an additive improvement of survival in preoperative CRT for rectal cancers and might overcome the hypoxic conditions of the tumor from the viewpoint of pathological local effect. However, this study showed the significantly positive correlation between HIF-1α expression and poor clinical outcome in the treatment of rectal adenocarcinoma, which indicates that the hypoxic tumors were still resistant to HCRT. Hence, it suggested that even addition of hyperthermia to preoperative radiation therapy could not entirely overcome the poor prognosis originated by hypoxia in the rectal cancers. There was no correlation between HIF-1α expression and pathological evaluation in the present study. Adenocarcinomas, which are colorectal cancer and prostate cancer, have a longer volume doubling time than squamous cell carcinoma. 36) Oka showed that adenocarcinomas had a low cycling cell population and showed no change during radiotherapy compared with squamous cell carcinoma. 37) Adenocarcinoma may require a long period to degenerate pathologically after radiation therapy compared with squamous cell carcinoma. Taking these reports into account, the histological assessment of the effect of radiation on rectal adenocarcinoma cells may require a longer period of observation for degeneration. In conclusion, this study suggests that the expression of HIF-1α is an important prognostic factor for preoperative chemoradiotherapy with hyperthermia in patients with rectal cancer. REFERENCES 1. Sauer R, et al (2004) Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med 351: Nelson H and Sargent DJ (2001) Refining multimodal therapy for rectal cancer. 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