Nakano_et_al-1993-Cancer.pdf

2401

Differential Values of Ki-67 Index and Mitotic Index of Proliferating Cell Population An Assessment of Cell Cycle and Prognosis in Radiation Therapy for Cervical Cancer

Takashi Nakano, M.D., and Kuniyuki Oka, M.D.

Background. Little is known about correlations between the growth fraction determined immunohistochemically with Ki-67 antibody and radiation response or prognosis after radiation therapy.

Methods. The prognostic value of the growth fraction determined by Ki-67 index and the mitotic index of proliferating cell population (pMI) were assessed in 45 cervical cancers treated with radiation therapy. The specimens from the cervix before radiation therapy were immunohistochemically stained with anti-Ki-67 antibody.

Results. The mean Ki-67 index and pMI for all patients were 36.0% and 2.74%, respectively. The patients with a Ki-67 index of 33% or greater showed significantly better histologic response to radiation at 30 Gy than those with less than 33%. The mean Ki-67 index for patients with good prognosis was significantly higher than for patients with tumor recurrence or metastasis later. Fur- ther, the mean values of pMI for patients with good prognosis were significantly lower than for patients with recurrence or metastasis. The 3-year survival rate for higher Ki-67 index (233%) was significantly better than lower Ki-67 index (less than 33%) (90.9% versus 34.8%; P < 0.001). However, the 3-year survival rate for higher pM1 (23.5%) was significantly poorer than lower pMI (less than 3.5%) (8.3% versus 81.8%; P < 0.001).

Conclusions. These results suggested that tumors with a high growth fraction showed a good prognosis with radiation therapy. In addition, the inverse prognostic correlation between the Ki-67 index and pMI suggested that both indices have independent values on radi-

From the Hospital Division, National Institute of Radiological Sciences, Chiba, Japan.

The authors thank Dr. K.R. Trott, Radiation Biology, London University, for manuscript preparation and suggestions and K. Ando, Ph.D., for comments.

Address for reprints: Takashi Nakano, M.D., Hospital Division, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage- ku, Chiba-shi 263, Japan.

Accepted for publication May 17, 1993.

ation response and prognosis after radiation therapy. Cancer 1993; 72: 2401-8.

Key words: Ki-67 index, growth fraction, mitotic index, prognosis, cervical cancer, radiation therapy.

Extensive efforts have been made to determine the prognosis of patients with various cancers after radiation therapy in terms of tumor proliferative parame- ter~. ' -~ Recently, the growth fraction has been determined immunohistochemically with the use of the Ki- 67 which recognizes a nuclear antigen expressed in all phases of the cell cycle except GO. Ki-67 index, which is a percentage of Ki-67-positive tumor cells, indicates a growth fraction and indicates a different nature of cell proliferation from the mitotic or labeling indices. We have already reported the difference between the growth fraction and mitotic index during radiation therapy for cervical cancer.' The percentage of Ki-67 index was highly variable and inversely correlated with prognosis in some turn or^.^^'^-'^ This was at- tributed to the association of the Ki-67 index with various characteristics of malignancy including nodal status, tumor grading, and labeling index. However, little is known about correlations between the growth fraction and radiation response or prognosis after radiation therapy. No significant correlation between the Ki- 67 index and prognosis has been reported in cervical cancer15 treated with preoperative radiation therapy or in prostatic cancer treated with hormonal therapy.16 Moreover, in radiation therapy, cancers with higher proliferative activity tend to possess higher radiation sensitivity.'' In contrast, tumor cells in the GO phase were reported to show radiation resistance." Hence, contrary to previous observations, the possibility remains that the tumor with highly proliferative cells indicated by a

2402 CANCER October 15, 1993, Volume 72, No. 8

Table 1. Ki-67 Index, Mitotic Index, and Mitotic Index of Proliferating Cell Population by Stage and Histologic Subtype

Ki-67 index Mitotic index PMI No. of patients Percent SD Percent SD Percent SD

Stage 1.9 & 0.41 1 2 69.6 f 22.9 1.37 f 0.72

2 8 30.0 k 5.9 0.67 f 0.33 2.34 +. 1.30 36.4 f 15.9 0 85 f 0.70 2.88 t 2.89 3 32

4 3 25 .8 f 2.7 0.73 f 0.32 2.87 +. 1.30

1.54 +_ 0.63 6.59 +. 2.69 1.90 2 1.76It ‘,t 2.59 t 1.60 2.74 +- 2.51

Kera tinizing 7 27.8 f 16.5 Large cell nonkeratinizing 31 38.3 f 14.2 0.67 f 0.54 Small cell 7 33.9 & 23.2 0.86 & 0.61

Total 45 36.0 f 16.1 0.83 f 0.63 SD: standard deviation; pMI: mitotic index of proliferating cell population. * P < 0.001. t P i 0.005. $ P i 0.01

Histologic subtype

I

high Ki-67 growth fraction may be sensitive to radiation and may show good prognosis.

The mitotic index of tumor cells in vitro usually is proportional to the cell proliferating speed, but the index of tumor cells in vivo is biased by the presence of quiescent cells whose population is larger than the cy- cling cell population in many cancer^.^,*',^^ Hence, it is important to assess proliferation speed and growth fraction of tumors separately. The mitotic index specifically of the proliferating cell population can express the relative cell cycle speed and can be estimated by the counting mitotic index and Ki-67 index as described below.

The purpose of the current study, therefore, was to determine the correlation between the Ki-67 or mitotic index and the histologic response to radiation therapy or prognosis, including failure patterns in radiation therapy for cervical cancer. In addition, the mitotic index of the proliferating cell population (pMI) is introduced and its value assessed for predicting the radiation response and determining prognosis.

Materials and Methods

Forty-five patients with invasive squamous cell carcinoma of the uterine cervix who received radiation therapy alone at National Institute of Radiological Sciences Hospital (Chiba, Japan) from 1988-1989 participated in this study. All patients were followed for more than 3 years. Clinical stages and histologic subtypes are sum- marized in Table 1. The clinical staging and histologic classification were based on the criteria of the Interna- tional Federation of Gynecology and obstetric^'^ and World Health Organization classifications.”

Radiation Therapy Protocol

Patients were treated with a combination of external and high-dose rate intracavitary irradiation. Details of the protocol were cited elsewhere.’l External whole pelvis irradiation was performed with anterio-posterior and posterio-anterior parallel opposed ports, with a dose of 1.8 Gy per fraction, five times per week, to a total dose of 30.6 Gy. This was followed by a central shielding pelvis field, with a dose of 2 Gy per fraction, five times per week, to a total dose of 20 Gy. Along with the central shielding irradiation, these patients also received intracavitary irradiation by remote afterloading system (RALS) using 6oCo sources. They received four insertions (one per week) with fraction dose of 5.5-6.0 Gy at point A, with the total doses were ranging from 22-24 Gy at point A.

Histopathologic Study

All specimens were excised from cervical tumors before and at 30 Gy during radiation therapy. All the fresh biopsy specimens were divided into two: one was fixed with 10% formaldehyde solution for conventional hematoxylin and eosin staining and the other specimen was quickly frozen for Ki-67 immunostaining. The specimens were cut with a cryostat in 6 pm thickness, air- dried, and fixed with cold 4% paraformaldehyde solution for 30 minutes. Then the sections were reacted with anti-Ki-67 monoclonal a n t i b ~ d y ~ , ~ (DAKO-PC; Dako, Copenhagen, Denmark) for 1 hour at room temperature. The sections were followed by reaction with biotinylated anti-mouse immunoglobulin G for 30 minutes and avidin-biotin complex2* (Vector Laboratories, Burlingame, CA) for 30 minutes. The sections were

Ki-67 Index and Modified Mitotic IndexlNakano and Oka 2403

reacted with 3,3'-diaminobenzidine tetrahydrochloride (DAB, Dojin Chemicals, Tokyo) solution with 0.01% (w/v) hydrogen peroxide23 for 2-5 minutes at room temperature and counterstained with hematoxylin. Control staining were done by incubating with phos- phate-buffered saline instead of anti-Ki-67 antiserum.

Assessment of Ki-67 Index and Mitotic Index

More than 1000 tumor cells per specimen were counted on three X200 color photographs for calculation of the Ki-67 index. The Ki-67 index was estimated by the percentage of Ki-67-positive cancer cells among all the counted tumor cells.

Counting mitotic figures is the traditional and most easily performed method for assessing cell proliferation. However, this method suffers from lack of stan- dardization and repr~ducibility.~~ To overcome these drawbacks in the current study, the mitotic index was assessed by counting 2000 cancer cells for each specimen from randomly selected fields, using three or more X200 color photographs of the hematoxylin and eosin staining.

Calculation of pMI

The mitotic index of tumors in vivo inevitably suffers from bias of the growth fraction of tumors. Hence, the index does not directly represent the proliferating activity of the cell population. The pMI can be calculated by the mitotic index divided by the growth fraction as the Ki-67 index as shown in the equation below:

M M/(P + Q) - Mitotic index = = P/(P + Q) Ki-67 index

-

where M, P, and Q indicate the mitotic cell population, the proliferating cell population, and the quiescent cell population, respectively.

Assessment of Histologic Response to Radiation Therapy

Early radiation response was assessed with punch biopsy specimens at 30 Gy. Histologic evaluation of the radiation effect on cancer cells was specified in the tumor nests and was determined using the histologic grading system proposed by Shimosato et al. (Table 2).25 In the current study, radiation responses of Grade I and IIA, which represent no or a little morphologic

Table 2. Histologic Grades for Radiation Effect

Grade Histoloeic findines

I Characteristics are noted in tumor cells, but tumor structures are not destroyed. There is no defect in tumor nests as a result of the disappearance of tumor cells.

In addition to characteristic cellular changes, tumor structures have been destroyed as a result of the disappearance of tumor cells. However, a variable number of viable cells remains.

Destruction of tumor structures is mild: viable tumor cells are observed frequently.

Destruction of tumor structures is severe: few viable tumor cells are seen.

Significantly altered, presumably nonviable tumor cells are present singly or in small clusters, and few viable cells are seen.

No tumor cells remain in any section (local cure).

I1

A

B

111

IV

change of tumor cells, were classified as poor response. Grade IIB and greater were defined as the finding of marked degeneration, but still viable-looking cells in one-third or less of the tumor nests and were classified as good response.

Statistical Analysis

All results of the various indices were statistically analyzed with either the chi-square test or F-test. Correla- tions among the Ki-67 index, pMI, and mitotic index were analyzed with the F-test. The cumulative disease- free survival rates were statistically analyzed with Peto log-rank test.26

Results

Ki-67-positive staining substance was located only in the nuclei of cancer cells, especially in chromatins and nucleoli, which showed diffuse nuclear staining and dot staining (Fig. 1). Ki-67-positive cancer cells were observed specifically in the peripheral or middle areas of most of the keratinizing type (Fig. 1, top) and disper- sedly in cancer nests of most of the nonkeratinizing cell types (Fig. 1, bottom).

Forty-five patients who were diagnosed as squa- mom cell carcinomas participated in the current study. The Ki-67 index, mitotic index, and pMI are shown by stages and histologic subtypes in Table 1. The mean Ki-67 index for all patients was 36.0% (range, 12.2- 85.8%). The mean mitotic index was 0.83% (range, 0.01-2.45%). The mean pMI was 2.74% (range, 0.03- 10.16%). There was no significant correlation between


stages and the above three parameters. However, the keratinizing type showed significantly higher mitotic index and pMI than the nonkeratinizing cell types.

Histologic radiation responses at 30 Gy of radiation therapy are shown in Table 3. Twenty-four patients were examined for their histologic radiation response. The patients with a Ki-67 index of 33% or greater showed significantly better histologic response than those with less than 33%. However, the mitotic index and pMI showed no correlation to the response and, in fact, demonstrated an inverse tendency compared with the Ki-67 index.

Correlations of the Ki-67 index, mitotic index, or pMI and patterns of failure are shown in Figure 2, left, center, and right, respectively. The mean Ki-67 index of the patients with good prognosis was significantly

Table 3. Histologic Radiation Response by Ki-67 Index, Mitotic Index, and Mitotic Index of Proliferating Cell Population

Histologic response

Poor Good Total Pvalue

Ki-67 index (Yo)

< 33 t 33

< 1 7 7 14 t l 6 4 10

< 3.5 9 9 18 2 3.5 4 2 6

Mitotic index (YO)

NS

pMI (%)

NS

pMI: mitotic index of proliferatinn cell population; NS: not significant

higher than those with recurrence or metastasis (P < 0.01). However, the mitotic index and pMI were inversely associated with local control and prognosis. A similar correlation was also observed in 32 patients with Stage I11 disease (data not shown).

The cumulative 3-year disease-free survivals for all patients (including patients with Stage I11 disease) by Ki-67 index, mitotic index, and pMI are shown in Figure 3. The cut-off values of the patients compared were 33% for the Ki-67 index, 1% for mitotic index, and 3.5% for pMI. The 3-year survivals for the Ki-67 index of 33% or greater were significantly better than those below 33% (90.9% versus 34.8% for all, 88.2% versus 26.7% for Stage 111; P < 0.001) (Fig. 3, top left). The 3-year survival rates for mitotic index of 1% or greater were somewhat lower than those below 1% (50.0% versus 74.1% for all, 42.9% versus 72.2% for Stage 111; P < 0.06) (Fig. 3, right). The 3-year survival rates for patients with pMI of 3.5% or greater were significantly lower than those below 3.5% (8.3% versus 81.8% for all, 10.0% versus 81.8% for Stage 111; P < 0.001) (Fig. 3, bottom left).

Three-year cumulative local control rates were calculated using Ki-67 index, mitotic index, and pMI. The local control rate for the Ki-67 index of 33% or greater was significantly higher than that of less than 33% (95.5% versus 60.1%; P < 0.01). The local control rate for mitotic index of 1% or greater was significantly lower than that of less than 1% (57.7% versus 92.4%; P < 0.01)). The local control rate for pMI of 3.5% or greater was significantly lower than that of less than 3.5% (21.191~

PMI is shown in Figure 4. No correlation between the

Figure 1. (Top) Ki-67 immunostaining for keratinizing type of squarnous cell carcinoma of the cervix (original magnification XZOO). The Ki-67-positive cells are observed in relatively outer parts of the cancer nests. (Bottom) Ki-67 immunostaining for large cell nonkeratinizing type of squamous cell carcinoma of the cervix (original magnification X200). The Ki-67-positive cells are observed diffusely throughout the cancer nest.

93.8%; p < 0.001). Prognosis by the Ki-67 index and mitotic index or

Ki-67 and mitotic indices was noted, whereas a slight

Ki-67 Index and Modified Mitotic Index/Nakano and Oka 2405

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Tumor Status Figure 2. (Left) Correlation of Ki-67 index and patterns of failure after radiation therapy for cervical cancer. The mean Ki-67 index was significantly higher in patients with good prognosis than in those with recurrence ( P < 0.01) or metastasis ( P < 0.05). (Center) Correlation of mitotic index and patterns of failure after radiation therapy for cervical cancer. The mean mitotic index was significantly lower in patients with good prognosis than in those with recurrence (P < 0.01) or metastasis (P < 0.05). (Right) Correlation of pMI and patterns of failure after radiation therapy for cervical cancer. The mean pMI was significantly lower in patients with good prognosis than in those with recurrence ( P < 0.001) or metastasis ( P < 0.001).

trend of a pMI decrease with an increase in the Ki-67 index was observed (r = 0.4). Most of the patients with recurrence or metastasis had tumors with a low Ki-67 index and a high mitotic index or a high pMI. The dis- crimination of prognosis was more significant by functions of the pMI and Ki-67 index than by functions of the mitotic and Ki-67 indices.

Discussion

The current study demonstrated that the Ki-67 index was positively correlated with prognosis and early radiation response in radiation therapy for cancer of the cervix. This contradicts the findings in lymphoma,' breast cancer,12,13 and soft tissue sarcoma,I4 where the Ki-67 index was inversely correlated with prognosis. The results of other authors were explained by the hy- pothesis that the higher proliferative activity of cancer cells shows a more malignant nature of the disease and results in a high frequency of recurrence or metastasis. However, no significant correlation has been reported in the outcome of cervical cancer,I5 where the results

were not conclusive because of the inconsistent background of the patients and the small number of patients studied. Prostatic cancer after hormonal therapy also showed no correlation.16

However, a more complex prognostic value of the Ki-67 index was observed in non-Hodgkin disease," where a higher Ki-67 index was associated with good prognosis for high-grade malignancy but with poor prognosis for low-grade malignancy. The current study may suggest that the proliferating cells measured by Ki-67 immunohistochemistry possessed high radiation sensitivity and, consequently, the tumors were easier to eradicate. Many investigations26-28 have suggested that GO cells, which are the major component of tumors with a low growth fraction, were resistant to radiation. One of the reasons is thought to be the higher repair activity of GO cells from potentially lethal damage caused by irradiation. Another possibility is that tumors with a low growth fraction are associated with hypoxic tumor cells, and the radioresistant hypoxic cell com- partment results in low local control characteristics.

The proliferative activity determined by the mitotic index showed a negative correlation with prognosis in


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the current study. A similar correlation had been reported in other cancers."-'4 However, this appears to contradict the positive prognostic value of the Ki-67 index. In addition, no correlation between the Ki-67 and mitotic indices was noted in the current study,

%

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10 20 30 40 50 60 70 80 90 100 Ki-67 Index

Figure 3. (Top left) The cumulative disease-free survival rates by Ki-67 index for all and Stage 111 patients. Patients with a Ki-67 index of 33% or greater showed significantly better survival than those with less than 33% in both all and Stage 111 patients (P < 0.01 for both groups). (Right) The cumulative disease-free survival rates by mitotic index for all and Stage 111 patients. The patients with a mitotic index of 1% or greater showed significantly poorer survival than those with less than 1% in all patients ( P < 0.05). A similar trend was observed in Stage 111 patients (P < 0.06). (Bottom left) The cumulative disease-free survival rates by pMI for all and Stage 111 patients. The patients with pMI of 3.5% or greater showed significantly poorer survival than those with less than 33% in both all and stage 3 disease ( P < 0.001 for both groups).

whereas a positive correlation between the Ki-67 index and the mitotic or labeling indices had been observed in other cancer^.^,^^-^^ Moreover, our previous study demonstrated different changes that the Ki-67 and mitotic indices during radiation therapy for cervical cancer

%

l o 1 C

0 Cont Rec Meta

Q"" .... 0 ..... 1 %

10 20 30 40 50 60 70 80 90 100 Ki-67 Index

Figure 4. (Left) Correlation among prognosis, Ki-67 index, and mitotic index. There was no significant correlation between Ki-67 index and mitotic index (r = 0.1; Y = 0.69 + 0.0046X). (Right) Correlation among prognosis, Ki-67 index, and mitotic index in proliferating cell population. There was a weak correlation between Ki-67 index and pMI (r = 0.4; Y = 5.14 - 0.065X).

Ki-67 Index and Modified Mitotic Index/Nakano and Oka 2407

were often disparate.’ These results suggest that the Ki-67 and mitotic indices are not always interdepen- dent.

Growth fraction and cell cycle time are independent major tumor proliferation characteristics. Gener- ally, the mitotic index, labeling index after thymidine labeling or bromodeoxyuridine incorporation, and the potential doubling time are used for accumulating information on cell kinetics. These proliferation indica- tors assess gross proliferation activity mixed with the growth fraction and cell cycle speed of the tumors, but they can not derive information from these two proliferative activities separately because these indices do not differentiate the involvement of the GO cell fraction.

The current study introduced the pMI to solve these problems. pMI excludes the quiescent cell population by use of Ki-67 immunostaining. High pMI suggested a shorter cell cycle time of the proliferative cell population, as the mitotic phase duration tends to be rather constant compared with other cell cycle phases.29 In the current study, high pMI indicated a significantly poor prognosis and was of greater prognostic reliability than the mitotic index. Hence, the faster cell production rate of the critical tumor cell population suggested by the high pMI may be one of the major causes of recurrence or metastasis. With regard to local recurrence, this might be due to fast repopulation of surviving cells. As for metastasis, however, it might be related to a higher risk of tumor cell invasion into the lymphatics by the more aggressively proliferating tumor cells.30

The current study indicated that tumors of the keratinizing cell type possess a rather high pMI and an average or somewhat lower growth fraction. Similarly, in head and neck cancer,31 well-diff erentiated squamous cell carcinoma (such as verrucous carcinoma) showed a high labeling index, short potential doubling time, and high cell loss factor. Therefore, contrary to the common perception, differentiated tumor may have a higher cellular proliferative activity than some undif- ferentiated tumors.

Sasaki et aL3‘ and Kame1 et al.33 reported that the labeling index in the proliferating cell population could be obtained by dividing it by the Ki-67 index for various cancers. They observed a wide variety in the labeling index of the proliferating cell population. These methods, however, require the administration of a toxic agent in patients or culture of excised tumor cells, and the labeling index would be affected by the perfusion activity under either condition. Additionally, no assessment has been made in respect to the clinical value of these indices. Both the Ki-67 index and pMI can be obtained directly from specimens from patients without carrying out any other procedures.

Recent studies on tumor proliferation during radiation therapy suggested that r e p ~ p u l a t i o n ~ ~ , ~ ~ or accelerated p r ~ l i f e r a t i o n ~ ~ , ~ ~ of surviving tumor cells reduced the chances of local control by radiation therapy. It is, however, not easy to measure these phenomena under routine conditions. In cancer clinics, then, the Ki-67 index and pMI may serve as effective predictive parameters for potential proliferative activity of tumors and are expected to contribute to the individualization of radiation therapy.

References

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Sasaki T, Sakka M. Implications of thymidine labeling index in the growth kinetics of human solid tumors. ]up ] Cancer Res

Begg AC, Hofland I, Moonen L, Bartelink H, Schraub S, Bon- temps P, et al. The predictive value of cell kinetic measurements in a European trial of accelerated fractionation in advanced head and neck tumors: an interim report. Int ] Radiat Oncol Biol Phys 1990; 19:1449-53. McNally NJ. Can cell kinetic parameters predict the response of tumours to radiotherapy? Int ] Radiaf Oncol B i d Phys 1989;

Dyson JED, Joslin CAF, Rothwell RI, Quirke P, Khoury GG, Bird CC. Flow cytofluorometric evidence for the differential radiore- sponsiveness of aneuploid and diploid cervix tumours. Ra- diother Oncol 1987; 8:263-72. Rutgers DH, van der Linden PM, van Peperzeel HA. DNA-flow cytometry of squamous cell carcinomas from the human uterine cervix: the identification of prognostically different subgroups. Radiother Oncol 1986; 7:249-58. Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Znt ] Cancer 1983; 31:13-20. Gerdes J, Lemke H, Baisch H, Wacker H, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. ] Im- rnunol 1984; 133:1710-5. Grogan TM, Lippman SM, Spier CM. Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-

Nakano T, Oka K. Transition of Ki-67 index of uterine cervical tumors during radiation therapy. Immunohistochemical study. Cancer 1991; 68:517-23. Hall PA, Richards MA, Gregory WM, dArdenne AJ, Lister TA, Standsfeld AG. The prognostic value of Ki-67 immunostaining in non-Hodgkin’s lymphoma. ] Pathol 1988; 154:223-36. Yamada Y, Murata K, Kamihira S, Atogami S, Tsukasaki K, Sohda H. Prognostic significance of the proportion of Ki-67 positive cells in adult T-cell leukemia. Cancer 1990; 67:2605-9. Wintzer H-0, Zipfel I, Schulte-Monting J, Hellerich U, von Kleist S. Ki-67 immunostaining in human breast tumors and its relationship to prognosis. Cancer 1991; 67:421-8. Stefan0 DD, Mingazzini PL, Scucchi L, Donnetti M, Marinozzi V. A comparative study of histopathology, hormone receptors, peanut lectin binding, Ki-67 immunostaining, and nucleolar or- ganizer region-associated proteins in human breast cancer. Cancer 1991; 67:463-71. Ueda T, Aozasa K, Tsujimoto M. Prognostic significance of Ki- 67 reactivity in soft tissue sarcomas. Cancer 1989; 63:1607-ll.

1981; 721181-8,

56:777-86.

67. Blood 1988; 71:1157-60.


15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Cole DJ, Brown CD, Crossley E, Alcock CJ, Gatter KC. Carci- noma of the cervix uteri: an assessment of the relationship of tumour proliferation to prognosis. Br ] Cancer 1992; 65:783-5. Raymond WA, Leong AS-Y, Bolt JW, Milios J, Jose JS. Growth fractions in human prostatic carcinoma determined by Ki-67 immunostaining. ] Pafhol 1988; 156:161-7. Frindel E, Tubiana M. Radiobiology and cell cycle. In: Baserga R, editor. The cell cycle and cancer. New York: Dekker, 1971:389- 447. Wallen AC, Ridinger DN, Dethlefsen LA. Heterogeneity of X- ray cytotoxicity in proliferating and quiescent murine mammary carcinoma cells. Cancer Res 1985; 45:3064-8. International Federation of Gynecology and Obstetrics. Annual report on the results of treatment in carcinoma of the uterus, vagina and ovary. vol. 16. Sweden: Radiumhemmet, 1979. Poulsen HE, Taylor CW, Sobin LH. Histological typing of fe- male genital tract tumors. no. 13. Geneva: World Health Organi- zation, 1975. Arai T, Nakano T, Morita S, Sakashita S, Nakamura YK, Fuku- hisa K. High dose rate remote afterloading intracavitary radiation therapy for cancer of the uterine cervix: a 20-year experi- ence. Cancer 1992; 69:175-80. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. ] His- tochem Cytochem 1981; 29:577-80. Graham RC, Karnovsky MJ. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new tech- nique. ] Histochem Cytochem 1966; 14:291-303. Clinical application of morphologic and immunocytochemical assessments of cell proliferation [editorial]. A m ] Clin Pathol

Shimosato Y, Oboshi S, Baba K. Histological evaluation of ef- fects of radiotherapy and chemotherapy for carcinomas. ]up ] Clin Oncol 1971; 1:19-35.

1992; 97:4-13.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Pet0 R, Pike MC, Armitage P. Design and analysis of random- ized clinical trials requiring prolonged observation of each pa- tient. B r ] Cancer 1977; 5:l-39. Rodriguez A, Alpen EL, Mendonca M, Deguzman RJ. Recovery from potentially lethal damage and recruitment time of noncy- cling clonogenic cells in 9L confluent monolayers and spheroids. Radiat Res 1988; 114:5151-7. Mendonca MS, Rodriguez A, Alpen EL. Quiescence in 9L cells and correlation with radiosensitivity and PLD repair. Radiat Res 1989; 117:433-47. Pardee AB. A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci U S A 1974; 71:1286-90. Tubiana M, Courdi A. Cell proliferation kinetics in human solid tumors: relation to probability of metastatic dissemination and long-term survival. Radiother Oncol 1988; 15:l-18. Dische S, Saunders MI, Bennett MH, Wilson GD, McNally NJ. Cell proliferation and differentiation in squamous cancer. Ra- diother Oncol 1989; 15:19-23. Sasaki K, Matsumura K, Tsuji T, Shinozaki F, Takahashi M. Re- lationship between IabeIing indices of Ki-67 and BrdUrd in human malignant tumors. Cancer 1988; 62:989-93. Kame1 OW, Franklin WA, Ringus JC, Meyer JS. Thymidine labeling index and Ki-67 growth fraction in lesions of the breast. A m ]

Tubiana MLH. Gray medal lecture: cell kinetics and radiation oncology. In t ] Radiat Oncol Biol Phys 1982; 8:1471-89. Maciejewski B, P-Bayer G, Trott KR. The influence of the number of fractions and of overall treatment time on local control and late complication rate in squamous cell carcinoma of the larynx. Znt ] Radiat Oncol Biol Phys 1983; 9:321-8. Withers HR, Taylor JMG, Maciwjewski B. The hazard of accelerated tumor clonogen repopulation during radiotherapy. Act On- cologica 1988; 27:131-46. Trott KR, Kummermehr J. Accelerated repopulation in tumours and normal tissues. Radiother Oncol 1991; 22:159-60.

Pathol 1989; 134~107-13.

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