Adjuvant inhibition of the epidermal growth factor receptor after fractionated irradiation of FaDu human squamous cell carcinoma

Radiotherapy and Oncology 72 (2004) 95–101 www.elsevier.com/locate/radonline

Adjuvant inhibition of the epidermal growth factor receptor after fractionated irradiation of FaDu human squamous cell carcinoma M. Krausea, F. Hessela, D. Zipsa, F. Hilbergb, M. Baumanna,c,* a

Department of Radiation Oncology, Medical Faculty Carl Gustav Carus, University of Technology Dresden, Fetscherstr, 74, 01307 Dresden, Germany b Boehringer Ingelheim Austria, Vienna, Austria c Experimental Centre, Medical Faculty Carl Gustav Carus, University of Technology Dresden, Fetscherstr, 74, 01307 Dresden, Germany Received 17 February 2004; received in revised form 7 May 2004; accepted 10 May 2004 Available online 20 May 2004

Abstract Background and purpose: Experiments performed by others have shown that inhibition of EGFR before and after single dose irradiation prolonged growth delay and improved local tumour control. This suggests that adjuvant EGFR inhibition can inactivate clonogens that survived irradiation. To test this hypothesis local tumour control was investigated after fractionated radiotherapy and adjuvant EGFR-TK inhibition. Materials and methods: FaDu hSCC xenografts were irradiated with 30 fractions in 6 weeks with total doses of 30 – 100 Gy. After the end of fractionated irradiation, BIBX1382BS was administered daily orally over a time period of 75 days. Tumour volumes were determined two times per week, the volume doubling time during adjuvant treatment was calculated for progressing and recurrent tumours. Local tumour control was investigated 120 days after end of irradiation. Results: Adjuvant BIBX1382BS significantly reduced the tumour growth rate but did not improve local tumour control. The TCD50 values were 66.1 Gy (95% C.I.: 59; 73 Gy) after adjuvant BIBX1382BS treatment and 67.9 Gy (61; 75 Gy) for control tumours ðP ¼ 0:9Þ: Conclusions: These data indicate that, although growth of recurrent tumour cells after irradiation is dependent on the EGFR pathway, tumour cells retain their clonogenic potential despite of EGFR inhibition. The results imply also that a decreased tumour growth rate does not necessarily allow conclusions on enhanced inactivation of clonogenic cells when antiproliferative drugs are combined with radiation. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: BIBX1382BS; EGFR TK inhibition; Molecular targeting; Adjuvant treatment; Fractionated irradiation; Squamous cell carcinoma; Human tumour xenografts; Tumour growth delay; Local tumour control

1. Introduction The epidermal growth factor receptor (EGFR), which is overexpressed in many human tumours, plays an important regulative role in tumour growth [39] and can be activated after irradiation [8,31]. Activation of the EGFR has been shown to increase cellular radioresistance [16,37] and has been suggested to be an important stimulus for accelerated clonogen repopulation during radiotherapy [12,21,29,30]. Therefore the combination of inhibitors of the EGFR with radiotherapy is a promising experimental strategy in cancer treatment [17,18,28]. In previous experiments simultaneous application of the EGFR tyrosine kinase (TK) inhibitor BIBX1382BS during irradiation with 30 fractions over 6 weeks significantly prolonged growth delay of FaDu human * Corresponding author. 0167-8140/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.radonc.2004.05.003

squamous cell carcinoma in nude mice but did not improve local control [2]. Sequential treatment with EGFR inhibitors is another option to improve the effect of radiotherapy [19]. Nasu et al. found improved local control of A431 tumours when C225 anti EGFR antibody was administered before (one injection) and after (two injections) single dose irradiation. These data suggest that adjuvant EGFR inhibition may kill clonogenic tumour cells. Recurrences after fractionated radiotherapy with doses in the curative range originate often from only one or a small number of clonogenic tumour cells that survived in a heavily damaged tumour bed. It is conceivable that these few surviving cells are inactivated if the EGFR pathway is suppressed or if an important growth factor such as EGF is withdrawn. The present study therefore addresses whether adjuvant application of BIBX1382BS after fractionated radiotherapy improves local control of FaDu tumours.

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2. Materials and methods

2.4. Tumour irradiation

2.1. BIBX1382BS EGFR-tyrosine kinase inhibitor

Local irradiations were given under ambient conditions without anaesthesia to air breathing animals (200 kV X-rays, 0.5 mm Cu, at a dose rate of , 1.1 Gy/min). Up to five animals were irradiated simultaneously in specially designed jigs. For the treatments the mice were immobilised in a plastic tube fixed on a lucite plate. The tumour-bearing leg was held positioned in the irradiation field by a footholder distal to the tumour. Irradiation protocols were started either on Mondays or Thursdays, the median tumour volume at start of the irradiation was 122 mm3 (10 – 90 percentiles 101 –210 mm3). Thirty equal fractions were applied within 6 weeks to total doses of 30, 40, 50, 60, 72.5, 85, and 100 Gy. The animals were randomised over the experimental matrix in groups of four, aiming for about eight animals (range 5– 9 animals) for each of the seven dose levels in each schedule. Animals were excluded from analysis if the uncertainty of dose application, e.g. by retraction of the leg from the irradiation field, was . 6.7% (i.e. . 2/30 fractions). The tumour control assays include data obtained from a total of 126 irradiated tumours. In addition 28 unirradiated control tumours were treated with BIBX1382BS or carrier in parallel to the tumour control assays.

BIBX1382BS, an TK inhibitor of the EGFR [10,23] was supplied by Boehringer Ingelheim Austria (Vienna, Austria). In FaDu, a cell line devoid of ErbB4, BIBX1382BS acts as a specific EGFR TK inhibitor at the low doses which can be achieved in vivo [2]. For the experiments 6 mg BIBX1382BS were dissolved in 1 ml carrier (980 ml hydroxypropyl-beta-cyclodextrin and 20 ml 1 N HCl). Fifty milligram per kilogram bodyweight (b.w.) BIBX1382BS were applied orally (p.o.) by gavage daily, including weekends. Carrier without BIBX1382BS was given to the control mice. The adjuvant BIBX1382BS- or carrier treatment started 24 hours after the last irradiation fraction and was continued over 75 days. Additional groups of animals received the adjuvant treatment over 180 days after irradiation doses of 30, 40 or 50 Gy. 2.2. Animals The experiments were performed using 7– 14 week old male and female NMRI (nu/nu) mice from the specific pathogen-free animal breeding facility of the Experimental Centre of the Medical Faculty of the University of Dresden. The animal facilities and the experiments were approved according to the German animal welfare regulations. The microbiological status of the animals was regularly checked by the veterinarians of the facility. The animal rooms provided day light plus a 12 h light and 12 h dark electric cycle (light-on time 07.00 am) and a constant temperature of 26 8C and relative humidity of 50– 60%. The animals were fed a commercial laboratory animal diet and water ad libitum. To immunosuppress the nude mice further, they were whole-body irradiated 2 days before tumour transplantation with 4 Gy using 200 kV X-rays (0.5 mm Cu) at a dose rate of about 1 Gy/min. 2.3. Tumour FaDu is an established, p53 mutated [11] human hypopharyngeal squamous cell carcinoma line, kept in high passage by the American Type Culture Collection (Rockville, MD). In nude mice FaDu grows as an undifferentiated carcinoma with a volume doubling time (VDT) between 100 and 400 mm3 of , 4 d [3,25]. In extensive series of quantitative tumour transplantation and of radiation tumour control assays FaDu tumours have been shown to evoke no or only a very low level of residual immune reactivity in nude mice [1,40]. For the experiments small tumour pieces were transplanted s.c. into the right hindleg of anaesthetised mice.

2.5. Follow-up, determination of tumour volumes and tumour growth delay Animals were observed until the mean diameter of the untreated or recurrent tumours exceeded 12– 15 mm, until death or until day 180 after end of treatment. Animals that appeared to suffer were killed before reaching these endpoints. Tumour diameters were measured twice per week for the first 60 –90 days after irradiation and once per week thereafter. Tumour volumes were determined by the formula of a rotational ellipsoid p=6 £ a £ b2 ; where a is the longest and b is the perpendicular shorter tumour axis. Conversion of tumour volumes to tumour mass (mg) was performed by a calibration curve based on excision weights [33]. Recurrences were scored when the volume increased for at least three consecutive measurements after passing a nadir. Median tumour volumes and their 95% confidence intervals were calculated for each treatment arm and dose level as a function of time after start of treatment. Volume doubling times (VDT) after irradiation were calculated for each individual tumour that recurred or progressed. For this, all tumour volumes measured until day 75 after the end of irradiation or until death of the animal were fitted by an exponential regression line. Since no significant differences in median tumour volumes and VDTs were observed between the groups receiving BIBX1382BS over 75 days and 180 days, the data were pooled for VDT calculations.

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2.6. Dose –response curves for local tumour control and TCD50 values Tumour control rates at day 120 after end of irradiation were calculated for each dose group after correction for censored animals according to the method given by Walker and Suit [36]. A binary (cure/failure) model was used to fit the individual tumour control data. For this, those animals that survived less than 20 days were omitted from analysis ðn ¼ 3Þ; and other censored animals were counted as local controls. The results did not change in any meaningful way when animals censored between day 20 and 54 were counted as recurrences. The tumour-control probability (TCP) was modeled using the logit model TCP ¼ 1=½1 þ expð2f ðx; bÞ


ð1Þ

where x ¼ vector of covariates that define the treatment, b ¼ vector of parameters describing radiosensitivity of the tumours, and f is a (possibly nonlinear) function of these. Parameters were estimated using maximum likelihood as implemented in STATA 7.0 software (STATA Corporation, College Station, TX). Quoted confidence limits are asymptotic estimates from the results of the likelihood fits. Comparison of maximum likelihood fits was performed using the likelihood ratio test [9]. TCD50 at day 120 after end of irradiation and associated dose –response curve were determined from: f ðD; bÞ ¼ b1 ð1 2 D=b2 Þ where b1 is a constant and TCD50 ¼ b2 2.7. Statistics Comparisons between groups with the Mann – WhitneyU Test were performed using GraphPad Prism 3.03 (GraphPad Software, Inc., San Diego, USA). Ninety-five percent confidence intervals (CI) of median values were determined as given by Sachs [26]. P values , 0.05 were considered as significant.

3. Results Fig. 1 shows the median absolute volume of FaDu tumours at the end of fractionated irradiation, i.e. at the start of BIBX1382BS treatment, for the different treatment groups. The tumour volumes decreased with increasing radiation doses between 30 and 72.5 Gy. No systematic differences of the tumour volume were observed between the animals randomised to receive adjuvant BIBX1382BS or carrier. To evaluate the effect of BIBX1382BS, all tumour volumes were normalised to their individual tumour volume at the start of drug application (relative volume). Fig. 2 shows the time course of the median relative tumour volume during treatment with BIBX1382BS or carrier for unirradiated tumours (panel a) and for tumours previously

Fig. 1. Median volume of FaDu tumours at the end of fractionated irradiation, i.e. at the start of daily treatment with BIBX1382BS (X) or carrier (W). The tumours were irradiated with 30 fractions in 6 weeks to total doses of 30, 40, 50, 60, 72.5, 85 or 100 Gy.

irradiated with total doses of 30, 40, 50 or 60 Gy (panels b– e). In all groups BIBX1382BS treated tumours grew slower than control tumours. Consistent with these results, the median volume doubling times (VDT) were significantly longer in the BIBX1382BS treated groups compared to the control groups (Fig. 3 and Table 1). As a reflection of radiation damage to the tumour bed, i.e. the tumour bed effect [6,24,35], the VDTs of preirradiated tumours were longer than the VDTs of unirradiated control tumours when BIBX1382BS was not applied (P , 0:0001; Table 1). However, when BIBX1382BS was given, no difference was observed between the VDT of unirradiated tumours compared with tumours irradiated before start of drug treatment ðP ¼ 0:6Þ; i.e. no tumour bed effect was detectable. Fig. 4 shows the observed local tumour control rates and the calculated tumour control probabilities after fractionated irradiation with 30 – 100 Gy in an overall treatment time of 6 weeks followed by adjuvant treatment with BIBX1382BS or carrier. The TCD50 value of 66.1 Gy (95% C.I. 59; 73 Gy) Table 1 Median volume doubling times of FaDu tumours after start of daily treatment with BIBX1382BS (X) or carrier (W). The tumours were either unirradiated or irradiated with 30 fractions in 6 weeks before start of drug application. Locally controlled tumours were excluded, treatment groups with more than 50% locally controlled tumours are not listed Total dose Median VDT Enhancement factor P-value (Gy) (95% C.I.) during treatment with BIBX1382BS Carrier 0 30 40 50 60 a

20.7 (13; 26) n.d.a 21.4 (17; 41) 20.2 (16; 22) 20.7 (20; 26)

6.3 (4; 8) n.d.a 10.2 (7; 15) 9.3 (7; 11) 9.4 (8; 12)

3.3 n.d.a 2.1 2.2 2.2

,0.0001 – 0.0057 0.0012 0.0357

Since the average tumour volume at the end of irradiation with 30 Gy was 1200 mm3, volume doubling times could not be determined for this group.

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Fig. 2. Time course of the median relative volume of FaDu tumours after start of daily treatment with BIBX1382BS (X) or carrier (W). The tumours were either unirradiated (a) or irradiated with 30 fractions in 6 weeks with total doses of 30 (b), 40 (c), 50 (d) or 60 Gy (e) before start of drug application. Treatment groups with more than 50% locally controlled tumours are not shown. Error bars indicate 95% C.I.

for 75 days adjuvant BIBX1382BS treatment was not significantly different from the TCD50 value of 67.9 Gy (61; 75 Gy) obtained for control tumours ðP ¼ 0:9Þ: The last recurrence in the BIBX138BS treated group was scored at day 83 after end of irradiation, all other recurrences before day 53. In a further cohort BIBX1382BS was applied for 180 days after irradiation with 30, 40 or 50 Gy. None of the 14 tumours irradiated with 30 and 40 Gy were locally controlled, three out of eight tumours were controlled after

50 Gy (data not shown in Fig. 4). The last recurrence occurred at day 53 after end of irradiation. These data do not allow calculation of a dose response curve for local tumour control. When both BIBX1382BS treated groups were pooled, the TCD50 was 64.4 Gy (57; 71 Gy). Also this value is not significantly different from the TCD50 value of the control group ðP ¼ 0:7Þ:

4. Discussion Previously published results have shown a prolongation

Fig. 3. Volume doubling times (VDT) of individual FaDu tumours during daily treatment with BIBX1382BS (X) or carrier (W) after fractionated irradiation with 30 fractions in 6 weeks. Locally controlled tumours were excluded, treatment groups with more than 50% locally controlled tumours are not shown. Since the average tumour volume at the end of irradiation with 30 Gy was 1200 mm3, volume doubling times could not be determined for this group. Horizontal lines indicate median values of the treatment groups.

Fig. 4. Local control rates and calculated tumour control probabilities (TCP, —) obtained for FaDu tumours irradiated with 30 fractions in 6 weeks with total doses between 30 and 100 Gy. After the end of the irradiation course BIBX1382BS (X) or carrier (W) was applied daily for 75 days. Error bars indicate 95% C.I. of the TCD50 values.

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of growth delay in FaDu tumours treated with the specific EGFR-TK inhibitor BIBX1382BS alone or simultaneously with fractionated irradiation [2]. Local tumour control was not improved after simultaneous treatment compared with irradiation alone. The aim of the present investigation was to evaluate the efficacy of adjuvant treatment with BIBX1382BS after fractionated irradiation. Since the number of remaining clonogenic tumour cells after highdose irradiation is expected to be small and since the surrounding tumour bed is severely damaged by irradiation, it appears conceivable that inhibition of proliferation of clonogenic tumour cells after the end of radiotherapy may improve local tumour control. However, this hypothesis was not confirmed in the experiments reported here. As in our previous study [2], application of BIBX1382BS in unirradiated tumours resulted in a significant reduction of the growth rate (Fig. 1, Table 1), confirming an antiproliferative effect of this drug in FaDu tumours. When BIBX1382BS was applied adjuvantly after fractionated irradiation the VDT of progressing tumours and recurrences increased significantly by a factor of two compared to tumours that were only irradiated. The magnitude of this effect was not different for the different dose levels applied before start of adjuvant BIBX1382BS treatment and did not depend on the tumour volume at start of drug application (Figs. 1 and 3). The results are in agreement with several other studies showing a reduction of the tumour growth rate and increased tumour growth delay when the irradiation is combined with adjuvant or partly adjuvant application of inhibitors of the EGFR. Milas et al. [21] reported enhanced tumour growth delay after the application of the anti EGFR antibody C225 6 h before and 3 and 6 days after single dose irradiation of A431 squamous cell carcinoma. The same observation was made by Saleh et al. [27] when eight injections of C225 were given after three fractions of 5 Gy in the same tumour model. Again in A431 tumours, Solomon et al. [32] found a decreased tumour growth rate when the EGFR tyrosine kinase inhibitor ZD1839 (Iressa) was applied during and after irradiation with four fractions of 2.5 Gy. It is interesting to note that a tumour bed effect, i.e. a decreased growth rate of tumours growing in preirradiated tissue [6,35], was only observed when BIBX1382BS was not applied. Furthermore the enhancement factor for BIBX1382BS treatment determined from the volume doubling times was 3.3 in unirradiated tumours but on average only 2.2 in irradiated tumours. This might indicate that the antiproliferative effect of EGFR inhibition is less in preirradiated tumours, e.g. as a consequence of impaired drug delivery to the surviving cells or due to changes of receptor expression. Alternatively, it may be speculated that part of the effect of EGFR inhibition on volume doubling in unirradiated tumours is caused by inhibition of neoangiogenesis [13]. This effect may possibly no longer contribute to reduced tumour growth after irradiation since irradiation in its own is a potent inhibitor of neoangiogenesis [4,7,20].

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Despite the significant prolongation of growth delay, adjuvant BIBX1382BS treatment had no impact on local control of FaDu tumours after fractionated irradiation. This indicates that inhibition of the proliferation after the end of radiotherapy via the EGF receptor does not result in additional clonogenic cell inactivation in this tumour. Only one other study so far investigated local tumour control after a combination of irradiation and, partly, adjuvant EGFR inhibition. In this study by Nasu et al. [22] either a single administration of the anti EGFR antibody C225 was given before single dose irradiation or three injections of the antibody were given 6 h before and 3 and 6 days after single dose irradiation. Both schedules improved local tumour control in A431 tumours, however the reduction of the TCD50 value was only significant after three applications of C225. These data suggest that adjuvant EGFR inhibition in A431 tumours may kill clonogenic cells. However, in contrast to the study presented here, one of three injections of the antibody was given before irradiation so that interactions between the effect of the first drug injection and radiation cannot be ruled out. Therefore no firm conclusions on the effect of adjuvant EGFR inhibition can be drawn from this study. Furthermore, tumour-tropism and efficacy of anti EGFR antibodies and tyrosine kinase inhibitors to kill clonogenic cells might be different, and A431 tumours, which have a very high expression of EGFR [38], might be more susceptible to EGFR related cytotoxic effects than FaDu tumours. All three studies that so far compared different experimental endpoints after combination of irradiation with EGFR inhibitors found more pronounced effects for tumour growth delay than for TCD50. Enhancement factors of 1.6 for one C225 injection and 3.6 for three C225 applications combined with single dose irradiation in A431 tumours were determined by Milas from growth delay data [21], while the dose modifying factors derived from TCD50 assays were 1.2 and 1.9 [22]. In our previous and the present study on FaDu tumours EGFR inhibition prolonged tumour growth rate and growth delay but this did not translate in improved local tumour control after fractionated irradiation. Tumour growth delay depends on the inactivation and regrowth rate of both clonogenic and non-clonogenic cells. Because of the larger proportion of non-clonogenic cells [1], tumour growth delay appears more likely to reflect the treatment effects on this cell population, whereas TCD50 depends solely on the inactivation of clonogenic cells [2,34]. It therefore appears preferable to the authors to use local tumour control as experimental endpoint when the effect of combined irradiation with antiproliferative substances on clonogenic tumour cells survival is investigated [15]. Alternatively, large growth delay studies that carefully evaluate several dose levels, and that correct for differences of growth rate in different experimental arms [5], may yield results as reliable and as relevant as tumour control assays [14,15].

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In conclusion, when given after the end of fractionated irradiation, the EGFR-TK inhibitor BIBX1382BS decreases the growth rate of FaDu tumours but does not improve local tumour control. These data indicate that, although growth of recurrent tumour cells after irradiation is dependent on the EGFR pathway, tumour cells retain their clonogenic potential despite of EGFR inhibition. The results imply also that a decreased tumour growth rate does not necessarily allow conclusions on enhanced inactivation of clonogenic cells when antiproliferative drugs are combined with radiation.

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Acknowledgements The excellent technical assistence of Mrs D. Pfitzmann, Mrs M. Oelsner, Mrs S. Balschukat and Mrs L. StolzKieslich, is gratefully acknowledged. The authors wish to thank Dr vet. P. Nelz, Dr vet. R. Kumpf and C. Geipel and their team for breading and maintenance of high quality nude mice. This work was supported by the Gerhard HessProgramm awarded by the Deutsche Forschungsgemeinschaft (Ba 1433/2).

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