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2280 Analysis of Response to Radiation Therapy of Patients with Cervical Adenocarcinoma Compared with Squamous Cell Carcinoma M I 6 4 and PClO Labeling Indices Kuniyuki Oka, M.D.'" Takashi Nakano, M.D? Tanji Hoshi, M.D." ' Department of Pathology, Mito Saiseikai General Hospital, Mito, Ibaraki, Japan. Unit of Clinical Examinations, National Institute of Radiological Sciences, Chiba, Japan. Unit of Clinical Oncology, 2,3. Division of Radiation Medicine, Research Center of Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, Japan. Institute for Urban Studies, Tokyo Metropolitan University, Tokyo, Japan. This study was supported by a grant for the diagnostic study group of charged particle therapy project (National Institute of Radiological Sciences; NIRS, Chiba, Japan). The authors thank Mr. 0. Mrozekfor the English revision, Dr. A. Tsuboi for helpful suggestions and advice, Mr. R. Ohuchi (NIRS), Mr. Y. Naoi, Mrs. S. Koyamatsu, Mr. S. Nakamura, and Miss T. Tobita (Mito Saiseikai General Hospital) for excellent technical support, and Mrs. M. Sakurai (NIRS) for secretarial assistance. Address for reprints: Kuniyuki Oka, M.D.,Department of Pathology, Mito Saiseikai General Hospital, 3-3-10 Futabadai, Mito, lbaraki 31 141, Japan. Received December 18, 1995; accepted February 9, 1996. 0 1996 American Cancer Society BACKGROUND. The MIB-1 monoclonal antibody is a marker of cycling cells and the PClO monoclonal antibody is a marker of proliferating cell nuclear antigen in paraffin sections. This study was conducted to elucidate the difference in response to radiotherapy (RT) between cervical adenocarcinomas and squamous cell carcinomas, focusing on cell proliferation. METHODS. A total of 196 biopsy specimens taken from the cervical carcinomas of 14 consecutive patients with adenocarcinoma and 62 patients with squamous cell carcinoma before and after RT at doses of 9 and 27 Grays (Gy) were investigated for MIB-1 and PClO immunoreactivities. RESULTS. In adenocarcinomas, the mean MIB-1 labeling indices before and after RT at 9 and 27 Gy were 28%, 21%, and 26%, respectively, whereas the mean PClO labeling indices were 15%, 13%, and 14%, respectively. In squamous cell carcinomas, the mean MIB-1 labeling indices before and after RT at 9 and 27 Gy were 38%, 53%, and 26%, respectively, and the mean PClO labeling indices were 23%, 23%, and 11%, respectively. CONCLUSIONS. Cervical adenocarcinomas have a lower cycling cell population and their indices show no change during RT. Squamous cell carcinomas have a higher cycling cell population and show a transient increase of the MIB-1 cycling cell population at 9 Gy of RT. These findings suggest a difference in response to RT between adenocarcinomas and squamous cell carcinomas. Cancer 1996; 77:2280-5. 0 1996 American Cancer Society. KEYWORDS: radiation, cell cycle, MIB-1 , PC10, cervical carcinoma, immunohistochemistry. I n general, the prognosis of adenocarcinomas of the cervix is regarded as poorer than that of squamous cell carcinomas'z2;two reasons for this are poorer local control rate and high incidence of metastasis. The poorer local control rate is thought to be due to the lower radiosensitivity compared with squamous cell carcinomas. Although the nature and clinical characteristics of squamous cell carcinomas have been studied extensively, the features of adenocarcinomas of the cervix are not yet fully understood, due to their low incidence. The mechanism behind the low radiation sensitivity of adenocarcinomas is a major concern and requires further investigation. Recently, many types of molecules associated with the cell cycle, including proliferating cell nuclear antigen (PCNA), Ki-67 antigen, and p53 oncoprotein, have enabled us to elucidate the proliferative status of human The growth fractions, proliferating cell nuclear antigen Response to Radiation in Cervical CarcinornalOka et al. (PCNA), and mitotic indices of squamous cell carcinomas of the uterine cervix have been intensively investigated by several groups of researchers, including our groups,"" and a transient increase in growth fraction has been reported in relation to the radiation response of the tumor cell^.^^'" However, the cell cycle dynamics of adenocarcinomas of the cervix during radiotherapy (RT) have not been evaluated. Analysis of the cell cycle in adenocarcinomas may elucidate the mechanism responsible for its low radiation sensitivity. The purpose of the present study, therefore, was to assess the tumor proliferation status of adenocarcinomas of the uterine cervix before and during RT in comparison with that of squamous cell carcinomas. METHODS We investigated I 4 patients with cervical adenocarcinornas who received a single established RT protocol at the National Institute of Radiological Sciences Hospital (Chiba, Japan). Their ages ranged from 36 to 82 years (mean, 65 years). Five patients had Stage IIB disease, six had Stage IIIb disease, and three had Stage IVA disease. Six patients had adenocarcinoma of the endocervical type, 2 had endometrioid adenocarcinoma, and six had adenosquamous carcinoma. We also investigated 62 paiients with cervical squamous cell carcinomas who received RT. Their ages ranged from 35 to 83 years (median, 63 years). Three patients had Stage I disease, 17 had Stage IIB disease, 38 had Stage IIIb disease, and 4 had Stage 1Va disease. Thirteen patients had squamous cell carcinoma of keratinizing cancer, 36 had large cell nonkeratinizing cancer, and 13 had small cell nonkeratinizing cancer. Radiotherapy Protocol The patients were treated with a combination of external beam RT and high dose rate remote afterloading intracavitary RT." The external pelvis RT was administered as 10-megaelectron volt photon beams or cobalt-60 gamma rays, with a dose of 1.8 Gray (Gy) per fraction, 5 times per week. The intracavitary RT was delivered by a Ralstron system (Shimazu Corporation, Tokyo, Japan) which has high dose rate cobalt-60 sources, with 2-4 Ci as the intracavitary source. The details of the RT procedure have been described previou~ly.~~" Histopathologic Study Prior to RT, biopsy specimens were obtained for the pathologic diagnosis of cervical tumors of all 14 patients with adenocarcinomas. Subsequently, one or two biopsy specimens were obtained from the cervical tumors during the period of RT, 1 day after total whole-pelvis irradiation of 9 Gy (n = 9) and/or 27 Gy (n = 7). Specimens were also obtained from the 62 patients with squamous cell 2281 carcinomas for the pathologic diagnosis of the cervical tumors before RT. Subsequently, one or two biopsy specimens were obtained from the cervical tumors during the period of RT 1, day after radiation at 9 Gy (n = 62) and1 or 27 Gy (n = 42). The intervals between the initiation of radiation and the biopsy at radiation doses of 9 and 27 Gy were 8 and 22 days, respectively. A total of 196 specimens were fixed with 10%formaldehyde for approximately 24 hours, and embedded in paraffin for hematoxylin and eosin staining and MIB-1 and PClO immunostaining. MIB-1 antibody recognizes Ki-67 antigen in paraffin section^.^,^ The MIB-1 labeling index represents the cycling cell population, this being equivalent to the growth fraction. PCNA is a nuclear protein associated with the cell cycle and DNA synthesis. Anti-PC10 antibody recognizes PCNA in paraffin sections, and the PClO labeling index mainly represents the Sphase fraction." For MIB- 1 immunostaining, the sections were cut and placed on glass slides coated with 3-aminopropyltriethoxysilane (LS-3150; Silicon Chemicals, ShinEtsu, Tokyo, Japan), after which they were heated 8 times in a household microwave oven (Mitsubishi Electric System Ovenrange RO-3700, Tokyo, Japan), each time for 3 minutes at about 95 "C, in 0.01 M citrate buffer (2.1 g citric acid monohydrate/liter distilled water, pH adjusted to 6 with NaOH). There were regular 1-minute intervals between the heating periods, and at the end of the procedure the specimens were left to cool down for more than 1 hour.I3 The sections were then incubated with anti-MIB-1 (Immunotech International, Marseilles, F r a n ~ e )or ~ antiPClO (Novocastra Laboratories, Newcastle-upon-Tyne, England)5 monoclonal antibodies for either 2 hours at room temperature or overnight at 4 "C, followed by incubation with biotinylated antimouse immunoglobulin G for 30 minutes and with avidin-biotin complex reagent (Vector Laboratories, Burlingame, CA) for another 30 minutes. The sections were subsequently reacted with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Dojin Chemicals, Tokyo, Japan) solution and 0.01% (weight/ volume) hydrogen peroxide for approximately 5 minutes at room temperature, and counterstained with hematoxylin. Negative controls were obtained by substituting phosphate-buffered saline for the primary antibody. Assessment of MIB-1 and PC10 Labeling Indices The MIB-1 and PClO labeling indices are the percentages of MIB-1- or PC10-positive cancer cells, respectively. The immunostained sections were evaluated blindly to avoid bias. Three randomly selected representative fields were used to count the cancer cells to determine the indices. A minimum of 1000 cancer cells (range, 1000 to 1500 cells) in every case was counted in 3 color photographs (X350) to minimize variations originating from heteroge- 2282 CANCER June 1,1996 I Volume 77 / Number 11 FIGURE 1. Adenocarcinoma cells are positive for MIB-1 (a) and PClO (b), showing intranuclear staining (MIB-1 and PCIO irnmunostaining, x350). neity within the sections. Cell counting was performed by the same observer (a pathologist), who was blinded to the identity of the patient from whom the specimen was obtained. Reproducibility was determined by counting in the same color photographs (twice in some cases) and by counting in different color photographs taken from other fields in the same cases. Variations between the first counting and the second and variations among the different fields were less than 5%. Statistical Analysis Statistical analysis was performed by the chi-square test and Student’s t test, as appropriate, using a computerized statistical package (HALBAU, Gendai Sugakusha, Tokyo, Japan). RESULTS MIB-1 and PClO antigens were present in the nuclei of cancer cells in both the adenocarcinomas (Figs. l a and b) and the squamous cell carcinomas (Figs. 2a and b).6,8 The mean MIB-1 and PClO labeling indices before RT in relation to the clinical stage and histologic subtype in the 14 patients with adenocarcinomas are shown in Table I, whereas Table 2 shows these results for the 62 patients with squamous cell carcinomas. There were no significant relationships between stage or subtype and the two indices in either group of patients. In patients with adenocarcinomas, the mean MIB- 1 labeling indices before RT and at 9 and 27 Gy were 28%, 21%, and 26%,respectively, and the mean PClO labeling indices were 15%, 13%, and 14%, respectively (Table 3). The mean MIB-1 and PClO labeling indices of the adenocarcinomas showed no remarkable change at 9 Gy or at 27 Gy (Figs. 3 and 4). In patients with squamous cell carcinomas, the mean MIB- 1 labeling indices before RT and at 9 and 27 Gy were 38%, 53%, and 26%, respectively, and the mean PClO labeling indices were 23%, 23%, and 11%, respectively (Table 3). The mean MIB-1 labeling indices of the squamous cell carcinomas were significantly increased at 9 Gy ( P < 0.01) and decreased at 27 Gy ( P < 0.01; Fig. 3). The mean PClO index did not show any remarkable change at 9 Gy and was significantly decreased at 27 Gy ( P < 0.01; Fig. 4). Table 3 demonstrates significant differences in MIB-1 and PClO indices between adenocarcinomas and squamous cell carcinomas before RT and at 9 Gy. However, at 27 Gy, the indices for adenocarcinomas and squamous cell carcinomas had similar values. DISCUSSION The present study showed that the mean MIB-1 and PClO labeling indices of adenocarcinomas were significantly lower than those of squamous cell carcinomas ( P < 0.001 and P < 0.001, respectively; Table 3). A similar cell cycle trend was observed in pulmonary carcinomas. Gatter et al.,I4in their investigation of 90 patients with pulmonary carcinoma (27 with adenocarcinomas and 63 with squamous cell carcinomas), reported that the Ki-67 labeling indices were lower in the adenocarcinomas than in the squamous cell carcinomas. Kawai et al.lS reported that the mean Ki-67 labeling index of pulmonary adenocarcinomas was 33%, whereas that of squamous cell carcinomas was 45%. Carey et reported that the mean PCNA labeling indexes of pulmonary adenocarcinomas and squamous cell carcinomas were 19% and 32%, respectively. These findings, as well as those in the present study, suggest that the proliferative activity of adenocarcinomas may be lower than that of squamous cell carcinomas, at least in terms of the percentage of the cycling cell population. The present study demonstrated differential changes in the MIB-1 and PClO indices of tumor cells between Response to Radiation in Cervical Carcinoma/Oka et al. 2283 FIGURE 2. Squarnous cell carcinoma cells are positive for MIB-1 (a) and PClO (b), showing intranuclear staining (MIB1 and PClO imrnunostaining x 350). TABLE 1 Mean MIB-1 and PClO Indices before Radiotherapy in 14 Patients with Cervical Adenocarcinoma in Relation to the Clinical Stage and Histologic Subtype TABLE 2 Mean MIB-1 and PClO Indices before Radiotherapy in 62 Patients with Cervical Squamous Cell Carcinoma in Relation to the Clinical Stage and Histologic Subtype Mean value pm SD (%b) Mean value 2 SD (W) Stage 2b 3b 3a Subtype EC .4S EM 'Total n MIB-1 index PClO index 5 6 3 28 2 4 26 i ti 33 i ti 14 2 7 16 2 3 15 2 2 ti ti 2 14 28 i ti 28 i 3 29 2 11 28 -C 6 14 i16 i15 I 15 5 4 7 2 5 iD: standard deviation: n: number of patients; EC: adenocarcinorna, endocenical type; EM: endometriadenocarcinoma; AS: adenosquamous carcinoma. 3id adenocarcinomas and squamous cell carcinomas during the period of RT. The MIB-1 labeling index of the squamous cell carcinomas significantly increased at 9 Gy and then decreased to a lower level than before RT at 27 Gy. However, the indices of the adenocarcinomas did not show any remarkable increase during the period of RT. To our knowledge, there has been no report to demonstrate differences in cell cycle changes between adenocarcinomas and squamous cell carcinomas during RT. It is not easy to explain this difference, because tumor cell kinetics during RT are considered to be associated with such complicated biologic factors at the tissue level as reoxygenation, repopulation, regeneration, and recruitment. One reason for the increase in the MIB-1 index of squamous cell carcinomas at 9 Gy, however, appears to be the re- Stage 1 2b 3b 4a Subtype LN SN K Total n MIB-I index PClO index 3 17 38 4 39 5 10 40 ? 14 37 i 14 43 i 17 35 ? 2 23 i 12 22 2 10 25 i 13 36 13 13 ti2 39 2 15 40 i 9 35 i 14 38 i 14 25 i 11 18 i 8 24 i I1 23 2 11 SD: standard deviation; n: number of patients: LN: large cell nonkeratinizing cancer; SN: small cell nonkeratinizine cancer: K keratinizine cancer. cruitment phenomenon, in which quiescent cells enter the cell cycle via the changes in nutritional or oxygenating circumstances induced by irradiation."'" Even if recruitment is one of the major associating factors in the early radiation period, it would seem to be less responsible for adenocarcinomas of the cervix. Opinions on the prognosis of cervical carcinoma in relation to histology are controversial. Some investigators have reported difference in survival rates between patients with cervical adenocarcinomas and those with squamous cell carcinomas," whereas others have reported that patients with cervical adenocarcinomas had a worse prognosis than those with squamous cell carcinomas. We have already reported that the 5-year survival rate of patients with Stage 111 2284 CANCER June 1, 1996 / Volume 77 I Number 11 TABLE 3 Mean MIB-1 and PClO Indices in Patients with Cervical Adenocarcinoma and Squamous Cell Carcinoma before and during Radiation Therapy Mean values ? SD (W Index Subtype Before RT 9 GY (n = 14) (n = 9) 27 Gy Ad (n = 7 ) P < 0.001 MIB-1 P < 0.001 sq (n = 62) (n = 62) Ad in 1 5=2 513) 1 (n = 41) (n = 8) P < 0.001 PClO P= 1 in = 7) P = 0.005 P = 0.451 sq (n = 62) (n = 62) (n = 42) S D standard deviation; before R T before radiotherapy; A d adenocarcinoma; Sq: squamous cell carcinoma; n: number of patients; Gy: Gray. P< 0.01.n=41 40 P<O.Ol, n=62 T 60 1” squamn-62 ........0.... adeno n-14 p<O.Ol, n=41 squam,n-62 T T 3.1 ....... 4-J” .”” adeno, n-14 10p=o.12, n=9 0- O i I p=O.55, n=7 I I 1 I 1 1 0 9 18 27 Gy Radiation dose FIGURE 4. Mean P C I O index with standard deviation before and during radiotherapy; 14 patients with adenocarcinornas ( . . . . .) and 62 patients with squamous cell carcinomas (-). cervical adenocarcinomas was 30%, poorer than that of patients with squamous cell carcinomas, which have a survival rate of 52%.”,” Similarly, the comparative radiation sensitivity of cervical adenocarcinomas and squamous cell carcinomas remains a subject of considerable controversy. Some investigators have suggested that the lower radiation sensitivity of adenocarcinomas explains the poorer local control rate. However, others believe that the poorer local control may be due to myometrial invasion rather than to lower radiation sensitivity.2o Recently, we demonstrated that adenocarcinomas of the cervix were rather radioresistant compared with squamous cell carcinomas when treated with high dose rate intracavitary radiation.‘l The present results indicate that the lower growth fraction in adenocarcinomas may be one reason for the radiation resistance of this tumor, be- cause a lower growth fraction indicates a large quantity of Go phase cancer cells, which are regarded as radiation resistant. The differences between adenocarcinomas and squamous cell carcinomas in the MIB-1 and PClO indices during RT may be associated with the differences in local control, although the differences in intracellular radiation sensitivity among various cell types are regarded as being responsible for radiation sensitivity. 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