Tumour cell kinetics as a prognostic factor in squamous cell carcinoma of the cervix treated with radiotherapy

Radiotherapy and Oncology 50 (1999) 77±84

Tumour cell kinetics as a prognostic factor in squamous cell carcinoma of the cervix treated with radiotherapy Anna GasinÂska a,*, Krzysztof UrbanÂski b, Jerzy Jakubowicz b, Mal¤gorzata Klimek b, Beata Biesaga a, George D. Wilson c a

Laboratory of Radiation Biology, Centre of Oncology, Garncarska 11, 31-115KrakoÂw, Poland b Department of Gynaecological Oncology, Centre of Oncology, KrakoÂw, Poland c Gray Laboratory Cancer Research Trust, Mount Vernon Hospital, London, UK Received 3 April 1998; received in revised form 5 July 1998; accepted 22 October 1998

Abstract Purpose: Proliferative rate and DNA ploidy status were evaluated by ¯ow cytometry in cervical cancer patients, prior to radiotherapy, to assess their importance as prognostic factors to predict survival rates. Material and methods: Between 1987 and 1995, a total of 260 patients with squamous cell carcinoma (SCC) of the cervix, FIGO stages IB±IIIB were analysed. Tumour samples were incubated with bromodeoxyuridine (BrdUrd) in vitro to measure their total labelling index (totLI) and LI (totLI for diploid and anLI for aneuploid tumours). Proliferation was also assessed by S-phase fraction (SPF) analysis of the DNA pro®le. Patients had intracavitary therapy (three applications, each of 16 Gy to point A) and XRT of 40±50 Gy given over 4±5 weeks. Results: The cervical tumours were characterized by a high proliferation rate which varied within each clinical stage of disease. The totLI ranged from 1.1 to 33.1% with median value of 9.6% whilst the LI ranged from 1.1 to 37.1% with a median value of 10.9%. Univariate analysis identi®ed patient's age (cut-offpoint # 50&greater; years) and to a lesser extent proliferation (cut-off point, median totLI ˆ 9:6%) as signi®cant prognostic factors for 5-year survival. The median survival time for younger patients ( # 50 years) with tumours of low proliferation (totLI # 9:6%) tumours was 17.5 months compared with 56 months in the faster proliferating tumours (P ˆ 0:0354). In the older patient sub-group, proliferation rate had no in¯uence on survival. The median LI value was not a useful parameter in survival. Cox multivariate analysis showed that patient age ( # 50 years) and low proliferation of the tumour cells (totLI # 9:6) were unfavourable prognostic factors for cervical cancers treated with radiotherapy. DNA ploidy was not signi®cant in this series. Conclusions: These data suggest that further improvements in therapy might be gained by selection of alternative treatments strategies such as neoadjuvant chemotherapy or radiation sensitizers in younger patients with more slowly proliferating tumours. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bromodeoxyuridine labelling index, DNA ploidy, prognostic factors, Squamous cell carcinoma of the cervix

1. Introduction Radiotherapy is administered to at least 50% of cancer patients with either curative or palliative intent. The majority of patients with squamous cell carcinoma (SCC) of the cervix will be treated with radiotherapy. However, the results of radiation treatment are not satisfactory, only about 39% of stage III patients will survive 5 years in Poland [2]. The effectiveness of radiotherapy is limited by both physical and biological parameters. Three of the most important biological parameters are considered to be proliferation, intrinsic radiosensitivity and hypoxia [30]. The * Corresponding author.

former is thought to be a major problem in conventional treatment schedules and the latter confers resistance on tumour cells treated with radiation. The clinical relevance of these parameters is currently receiving considerable attention [24,31,36]. The application of cell kinetics in cancer has a simple rationale, faster tumour proliferation may be associated with greater repopulation potential and a greater risk of failure with protracted treatments. Generally, it is assumed that fast proliferation of tumour cells is an unfavourable prognostic factor [1,4,6]. Therefore, detailed knowledge of individual tumour cell kinetics is important to assess prognosis and for potential use to individualize therapy [9,28]. Using ¯ow cytometry (FCM), knowledge of tumour proliferation can be gained from analysis of the incorpora-

0167-8140/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0167-814 0(98)00135-2

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A. GasinÂska et al. / Radiotherapy and Oncology 50 (1999) 77±84

tion of halogenated pyrimidines into DNA and from cell cycle analysis of the DNA pro®le. Both of these techniques use DNA content analysis which provides further information on the ploidy status of the tumour. In some malignancies patients with diploid tumours show a clear advantage in terms of survival suggesting that aneuploid tumours might need more aggressive treatment [13]. A drawback in FCM analysis of tumours is the inability to discriminate normal from neoplastic cells if the specimen is diploid, this leads to an underestimation of the LI [36]. In aneuploid tumours, this inaccuracy can be overcome by restricting analysis to those cells with abnormal DNA. However, this practice results in a biased analysis in which diploid tumours will, by default, be considered more slowly proliferating in comparison with aneuploid specimens. To overcome this, we have calculated two values for LI. Firstly, the total LI (totLI) which represents all cells in the specimen irrespective of DNA content; this provides an impartial comparison for all tumours. Secondly, we have used the term LI to indicate the best estimate of proliferation, i.e. the totLI for diploid tumours and the aneuploid LI (anLI) for aneuploid tumours. In Sphase fraction analysis, the same problems exist as the computer algorithm is ®tted to all cells in diploid tumours but is restricted to aneuploid cells in those tumours with abnormal DNA. The aim of the study was to assess proliferative potential (totLI, LI, S-phase fraction) and DNA ploidy in advanced SCC of the cervix and to study the in¯uence of these parameters on patient survival after radiotherapy. 2. Materials and methods 2.1. Patients The study consisted of 260 patients with SCC of the cervix treated with radiotherapy alone over the period of September 1987 to May 1995. Eleven patients had FIGO stage IB cancer, 100 patients had stage IIA, 85 patients had stage IIB, four patients had stage IIIA and 60 patients had stage IIIB. Patients' ages were similar in each stage and ranged from 27 to 79 years (median 54 years). 2.2. Material Between 1987 and 1989, ¯ow cytometric analysis (110 tumour samples) was carried out at the Gray Laboratory, and the remaining 150 tumours were analysed in the Centre of Oncology, KrakoÂw, Poland. 2.3. Irradiation Patients with SCC of the cervix in stages IB±IIA had three intracavitary applications each of 16 Gy to point A using 137Cs irradiation (Selectron LDR) separated by 4±5day intervals. After a 2±3week break, external beam irradia-

tion was administered, consisting of 40 or 50 Gy (in 20 or 25 fractions given over 4±5 weeks, fraction dose 2.0 Gy). In the higher stages, IIB±IIIB, the reverse schedule of radiotherapy was applied, external beam being applied prior to curietherapy. In addition, 46 of the 260 patients were treated twice daily for 16 days with a dose per fraction of 1.7 Gy separated by 6-h intervals, to a total dose of 54.4 Gy. After a 3week break, curietherapy was administered as described above. Overall treatment time for conventional treatment was between 56 and 62 days and 52 days for hyperfractionated radiotherapy. 2.4. BrdUrd labelling index In vitro incorporation of BrdUrd was carried out according to the high pressure oxygen method described by Steel and Bensted for tritiated thymidine [25]. The BrdUrd staining procedure and ¯ow cytometry has been described in detail elsewhere [34]. Brie¯y, after 1 h of incubation with BrdUrd at 378C, tumour fragments (about 0.1 cm 3) were ®xed in 70% ethanol. They were then minced into fragments and digested into nuclei at 378C with 0.4 mg/ml pepsin (Sigma, Poole, Dorset) in 0.1 M HCl for 20 min. The nuclei suspension was ®ltered through 35 mm nylon mesh and centrifuged at 2000 rev./min for 5 min. The pellet was resuspended in 2 M HCl for 12 min to denaturate the DNA partially. After two washes in phosphatebuffered saline (PBS), the pellet was incubated in PBS containing 0.5% normal goat serum (NGS) (Sigma), 0.5% Tween 20 (Sigma) and a mouse-derived anti-BrdUrd monoclonal antibody (Dako). After 1 h, the nuclei suspension was washed in PBS and suspended in PBS/NGS/ Tween containing goat anti-mouse IgG FITC conjugate (Dako) for 1 h. After two further washes in PBS, the suspension was stained for total DNA with PBS containing 10 mg/ml propidium iodide (PI). 2.5. Flow-cytometric data analysis The stained preparations were analysed with an Ortho Cyto¯uorograph in the Gray Laboratory and a FACScan ¯ow cytometer in the Centre of Oncology in KrakoÂw. Doublets and clumps were excluded from the stained preparations by gating on bivariate distributions of red peak versus area signal in the Ortho and FL3 area versus width signal in the FACScan. Excitation of the FITClabelled cells and the DNA-associated PI was accomplished with an argon-ion laser tuned to 488 nm and operated at 200 mW in the Ortho and using 15 mW in the standard FACScan con®guration. At least 10 000 events were collected in each specimen. The numbers of labelled S-phase cells (totLI) was calculated as the percentage of nuclei that incorporated BrdUrd. If the tumour was aneuploid, the LI was calculated for the aneuploid subpopulation (anLI) by exclusion of the diploid cells. The SPF was calculated using the sum of broadened rectangles model (SOBR) both for the Ortho and as part of the CellFIT

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79

3. Results 3.1. Proliferation, ploidy and clinical stage

Fig. 1. Heterogeneity in the totLI values within each stage of cervical cancer.

software package on the FACScan. As it was not possible to assess the duration of S-phase after in vitro BrdUrd labelling [5], a predicted Tpot (predTpot) was estimated (using the LI) and the formula Tpot ˆ l Ts/LI using a Ts value of 15.8 h based on previous studies of cervix tumours labelled in vivo [35]. The DNA index was calculated from the ratio of the modal DNA ¯uorescence of abnormal to normal G1/0 cells. Aneuploidy was assessed in cases in which the normal and neoplastic cell populations gave two separate peaks. Human lymphocytes were used as a reference peak. 2.6. Statistical analysis The probability of survival and failure was calculated by the Kaplan±Meier method [18]. Log-rank test was used to assess the relationship between actuarial survival and the assessed parameters. The cut-off point of 50 years for age and median totLI, median LI were used. Cox multivariate analysis [10] was performed with a forward stepwise procedure to select a set of signi®cant prognostic factors at the pre-set level a ˆ 0:05.

Cervical tumours showed great variability in proliferation characteristics with BrdUrd totLI's ranging from 1.1 to 33.1% with a median value of 9.6%. The median LI was slightly greater at 10.9% (range 1.1±37.1%). The median SPF was signi®cantly greater at a value of 24.9% but was highly correlated with totLI and LI (P&greater; 0:001). Using a ®xed value for Ts as described, the predicted median Tpot for the 260 patients would be 4.8 days (range 1.4±48.3 days). The analysis was simpli®ed to consider stages I, II and III (Fig. 1) as we have found no statistical difference between subgroups A and B in stages II and III. The median pretreatment totLI for stage I was 9.5%, 9.3% for stage II and 10.5% in stage III. The median LI for stage I was 9.5%, 10.4% for stage II and 11.7% for stage III. The predicted Tpot values were 5.5, 4.8, 4.7 days for stages I, II, and III, respectively. Median values for SPF did not differ between stages (25.5%, 23.2% and 27.9% for stages I±III). These results provide no evidence that proliferation increased with increasing stage. There was also no signi®cant difference in the percentage of tumours classi®ed as fast proliferating (on the basis of the median totLI value or LI) as a function tumour stage (P ˆ 0:3379). Patient age and proliferation were not correlated. The median totLI and LI were 10.2% and 9.3% and 11.7% and 10.4% for young ( # 50 years) and older patients ( . 50 years), respectively. The overall incidence of aneuploidy was 56% in this series with a modal DNA index value of 1.7. In seven tumours the mathematical model failed to assess the DNA index. There was no signi®cant difference in totLI between diploid (111 specimens) and aneuploid (142 specimens) tumours. However, as expected, the median LI (P ˆ 0:019) and SPF (P ˆ 0:015) were signi®cantly higher in aneuploid lesions. There was no evidence to suggest that the incidence of aneuploidy increased with tumour stage. 3.2. Proliferation, ploidy and clinical outcome The follow-up period ranged from 6 to 100 months with a median duration of 53 months; the survival rate was calculated with a 5-year cut-off. Of 260 patients 132 (50.8%) remained alive, 109 of them (41.9%) without evidence of disease. A total of 145 patients were locally controlled (Table 1). In univariate analysis, patient age, tumour stage, proliferation parameters and DNA ploidy were analyzed. Tumour stage was not signi®cant in this series of patients (P ˆ 0:2323). However, patient age (P ˆ 0:0147) and proliferation rate assessed by totLI (P ˆ 0:0457) appeared to have in¯uence on survival. LI, SPF and DNA index showed no signi®cance in univariate analysis.

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80

Table 1 Five-year follow-up for cervical patients treated with radiotherapy Endpoint

N

(%)

Alive with no evidence of disease Local control Alive with disease Died from ca or metastases Died from intercurrent disease Lost to follow-up

109

41.9

145 23 107 8 13

(59.4) 8.8 41.1 3.1 5.0

In this series, patients were strati®ed above and below 50 years of age, as this is the accepted cut-off for pre- and postmenopausal women in Poland. It should be noted that the median age of 54 years was not signi®cant in univariate analysis, although cut-off values between 41 and 52 years did show signi®cance. The median survival time for younger patients ( # 50 years) was shorter (32 months) than that of the older patients ( . 60 months, P ˆ 0:0147). Moreover, the probability of survival for younger patients ( # 50 years) with totLI # 9.6% was signi®cantly worse than those with higher proliferation indices (totLI . 9.6%, P ˆ 0:0354, Fig. 2a). The median survival time for the former group was 17.5 months and 56 months for the latter group. In the older patient sub-groups, proliferation parameters had no in¯uence on patient survival (P ˆ 0:2903, Fig. 2b). However, the median survival time was higher than that found in younger patients ( . 60 months). Neither median LI nor SPF had the power to predict survival in younger patients, P ˆ 0:093 and P ˆ 0:507, respectively. Patients with aneuploid tumours had signi®cantly worse local control than those with diploid tumours (P ˆ 0:0333) but tumour ploidy was without overall in¯uence on patient survival. However, subgroup analysis revealed that patients with fast proliferating ( . median totLI) aneuploid tumours survived signi®cantly longer (P ˆ 0:0423) than those with slowly proliferating tumours. None of the other proliferation parameters revealed differences in survival or local control even in the younger patient group. In multivariate Cox analysis, age, stage, SPF, totLI, LI, predTpot, DNA ploidy and schedule of radiotherapy were entered. Cox analysis showed that only patient age ( # 50 years) and low proliferation of the tumour cells assessed by totLI # 9:6% were unfavourable prognostic factors (Table 2). 4. Discussion Cell kinetic characteristics were evaluated in a cohort of 260 patients with cervical cancer and related to clinicopathological features and outcome. Prognostic markers which are required in cervix cancer as clinical features

such as tumour stage, were not shown to be useful parameters for prediction of survival in this study as has been found previously [20,24,27]. This might, in part, be due to the more advanced stage IIIB patients being treated by a higher total dose in the hyperfractionated treatment although the in¯uence of the schedule was not signi®cant using Cox analysis. However, patient age did appear to have signi®cant impact on survival, con®rming other studies [23,26,31]. However, the signi®cance of this parameter often depends on number of patients studied and their age distribution; it is dif®cult to show impact of age on survival in smaller series of patients or those including older patients [8,27,37]. We have used 50 years of age as the cut-off point based on the accepted division of pre- and postmenstration in Poland. The choice of cut-off in¯uences the signi®cance of the data, any value between 41 and 52 years resulted in a signi®cant P-value in univariate analysis whereas the mean age of 52 years or median age of 54 years failed to show any statistical signi®cance. Hormonal status in young patients might in¯uence tumor biology resulting in a more aggressive phenotype and might explain our earlier study [29] showing that adjuvant progestagen therapy improves survival in patients with endometrial cancer after hysterectomy. The results of cell kinetic parameters analysis performed in this study would suggest that cervical carcinoma represents a group of tumours capable of rapid proliferation. The median values for totLI and LI obtained in our study are within the range (4.8±12.0%) reported by other authors [7,12,15,19,21,37]. Some authors who used in vivo BrdUrd also received similar results [7,27]. We incorporated BrdUrd in vitro into small tumour fragments (about 1 mm 3) which means that if the diffusion distance of BrdUrd is similar to O2 (about 150 mm) all cells should be exposed to BrdUrd. However, no-one has really measured how far it will diffuse in the presence of high oxygen pressure as in the culture system. An earlier study [33] might suggest that in vitro and in vivo labelling does give comparable results. It should be also said that labelling is very heterogenous after in vivo labelling due to both erratic vasculature and tumour histology. Also the predicted Tpot range (1.4±48.3 days) correlates with other data obtained from direct measurement [7,27]. This study con®rms the general consensus that the LI is an independent parameter not correlated with patients' age, stage, DNA ploidy or tumour differentiation [12,20,34]. In this study we have attempted to redress the bias between diploid and aneuploid tumours by analysing both a totLI and the LI corrected for aneuploidy when appropriate. The totLI represents a complex parameter, it is a balance between the number of tumour cells present and their proliferation rate. The totLI could be low because of little tumour cell proliferation or because there are few tumour cells in the specimen. If the latter possibility is re¯ected in the cellular composition of the primary lesion, then the tumour may be more radiocurable, as there are

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Fig. 2. Survival as a function of totLI, strati®ed using the median totLI # 9:6%&greater;. Patients were divided into those below (a) and above 50 years (b). Fifty younger and 56 older patients died within 5 years follow-up.

fewer cells to kill. It was interesting to note that the totLI, and not the LI, was the only parameter to show statistical signi®cance in predicting outcome, particularly in younger patients. This might suggest that the practice of correcting the aneuploid LI, but not the diploid, only serves to confuse the potential prognostic information. We have found a high correlation (r ˆ 0:94) between LI of neoplastic cells and totLI in aneuploid tumours. Proliferative differences between diploid and aneuploid tumours, evident from

consideration of LI or SPF, were not apparent with totLI. This questions whether fundamental differences exist between diploid and aneuploid tumours. Indeed, several authors have assessed the prognostic value of DNA ploidy in cervical carcinoma and the results are contradictory. Some authors [3,22,38] reported worst survival for patients with diploid tumours whilst others conclude that these tumours have the most favourable outcome [13]. Yet, in other studies [11,14,16,17,26,38] no signi®cant

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82

Table 2 Cox multivariate factor analysis of survival of cervical cancer patients treated with radiotherapy (®nal model). TotLI represents number of total labelled S-phase cells in the analysed sample Model

Variable

RR a

P value

I

Age # 50 years . 50 years

1.73 1.00

0.0056

totLI # 9.6% . 9.6%

1.58 1.00

0.0211

a

Relative risk with 95% con®dence limits.

difference was observed between aneuploid and diploid tumours. In this study the incidence of aneuploidy of 56% was similar to that in other reports, 71% [7], 65% [23], 60% [38], and we did not observe a correlation with clinicopathological features or survival. However, patients with diploid tumours had signi®cantly better local control than those whose tumours were aneuploid. The in¯uence of proliferation on the outcome of cervix cancer has been studied by a variety of methods. Tsang et al. [27] dichotomized patients according to the median Tpot, and LI values and, in contrast to our data, showed that short Tpot values or high LI were weakly associated with shorter disease-free survival. However, their series consisted of only 46 patients and, unlike the present study, they were unable to show any in¯uence of age on survival. This may have been due to the preponderance of older patients (63% were greater than 50 years old). In the present study SPF was not predictive of outcome. This was also found by Tsang et al. [27] and Zolzer et al. [39]. The latter found that neither SPF nor the micronucleus frequency before radiotherapy could predict outcome. However, in their study, when changes in response to therapy were considered, patients whose SPF decreased and patients whose micronucleus frequency increased tended to have a better prognosis. The conclusion from this present study would be that tumours with a high proliferative index were more radioresponsive. Oka et al. [22] have recently revealed the difference in response between squamous cell carcinoma (SCC) and adenocarcinoma (AC) following radiotherapy in the cervix. Cervical AC contained a lower percentage of cycling cells than SCC and, in contrast to SCC, showed no change in the cycling cell population during radiation treatment. Temporary increases in the percentage of proliferating cells might indicate cell repopulation induced by cell depopulation as a result of radiotherapy. No change in the number of proliferating cells after treatment in AC might suggest a lack of radiation-induced cell loss and might help to explain the poorer radiotherapy results in AC in comparison with SCC observed in the clinic. The balance between cell turnover and cell loss is crucial in determining the response of tumours to treatment and

these two processes are likely to be closely coupled such that a high cell turnover in fast proliferating tumours may be associated with a high rate of apoptosis. Levine et al. [20] examined the percentage of apoptotic cells (apoptotic index, AI) in pre-therapy cervical biopsies and found that the 5year survival rate for tumours with an AI below the median was signi®cantly greater than the rate for those with an AI above the median. However, the authors were unable to show correlation of proliferation with outcome using Ki67 detection as have other studies [9]. However, contradictory results were obtained in other studies [32], who showed that higher pretreatment apoptosis in cervical adenocarcinoma was prognostic factor for patients survival. Favourable prognostic signi®cance of high tumour proliferation was suggested in earlier studies of cervix carcinoma. Dixon et al. [12] observed that the mean LI was higher in radioresponsive tumours, though not reaching statistical signi®cance. Courdi et al. [9] observed a signi®cant fall in LI after radiotherapy in patients with a poor outcome whereas the fall was not signi®cant or less signi®cant in patients who survived 5 years. In the Lagrange et al. study [19], the LI correlated with the histological ®ndings following laparotomy. Slowly proliferating cells were often observed in situ after tumour removal, presumably since they required a longer time to be eliminated. Recently, Zamulaeva et al [37] have shown better 3±6 months complete response rates and improved probability of 3year survival in patients with LI . 7:0% compared with those with LI , 7:0 % A possible explanation for these ®ndings has been that tumours with a low LI are more dif®cult to sterilize due to, either inherent radioresistance, or an association with an unfavourable condition such as hypoxia. West et al. [30,31] have con®rmed the ®rst hypothesis. Radioresistant cervical tumour cells are signi®cantly more dif®cult to treat with radiotherapy [31]. They have shown that the 5-year survival rate for tumours with SF2 values below the median (0.42) was 81% and was signi®cantly greater than rate of 51% for those with SF2 values above the median. In conclusion, it has been suggested that tumours with a high LI are more radioresponsive, but there had been no data available to show that they are more radiocurable. Our results and those of Zamulaeva et al [37] would suggest that the latter is also true in this group of cervical cancers. The reasons for this are unclear. Radiosensitivity may be improved by redistribution of cells into more sensitive phases of the cell cycle in the higher LI group. Alternatively, a high proliferation rate may correlate with a higher level of spontaneous and radiation-induced apoptosis [32]. The overall treatment time or strategy would appear to have little in¯uence in this series of patients although the improved response of the most advanced cervical tumours may have been due to the higher total dose applied to 20% of patients in the hyperfractionated treatment. The poorer response of younger patients with more slowly proliferating tumours indicates the need to consider other treatment

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options such as neoadjuvant chemotherapy or radiation sensitizers. Acknowledgements The authors thank Prof. Andrzej Sokol¤owski for statistical analysis of the data and Prof. Jan Skol¤yszewski for reviewing the manuscript. We are grateful to Mrs. Anna Cichocka for the excellent technical assistance. This study was partially supported by the International Atomic Energy Agency, Contract no. 7292/RB.

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