Animals were being observed up to ultimate tumour sizing (WP1066 imply diameter exceeded fifteen mm), till working day 150 right after the WP1066 end of treatment or until loss of life. Animals that died from tumour-independent reasons and therefore could not be followed up, were censored at the last day of measurement.When death occurred before day 20 after end of treatment animals (without recurred tumour) were excluded from the analysis. Animals with local failure before death were not excluded from the analysis but counted as failure. Based on the individual tumour control data a binary (cure/failure) model was used to fit tumour control probability (TCP) curves. The TCP was modeled using the logit model.
Tumour cells of the different cell lines were grown in Dulbeccos modified Eagle’s medium with glutamine, 10% fetal calf serum, 1 mM pyruvate, 1% non-essential amino acids, 20 mM HEPES and 1% Penicillin-Streptomycin at 37°C (5% CO2, 95% humidity) in 25 cm2 tissue culture flasks. For the A7 tumour model MEM-EARLE with 10% fetal calf serum and 1 mM pyruvate was used as medium. After 24 hours cells were incubated with BIBW 2992 or for control with dimethylsulfoxid (DMSO). Three days later, cells were irradiated with doses of 2, 4, 6 or 8 Gy. Directly after irradiation (200 kV x-rays, 0.5 mm Cu, ~1 Gy/min) cells were trypsinised and counted. Appropriately diluted single cell suspensions were incubated in petri dishes for 14 or 10 days (A431), fixed and stained with crystal violet.Colonies with ≥ 50 cells were scored as survivors. Mean values of the surviving fraction and their standard deviations (SD) were determined for each treatment group and dose level. Cell survival curves were fitted according to the LQ-model. Plating efficiencies (PE) and surviving fractions (SF) were calculated using the following formulas:
Figure 2 shows the relative tumour volume as a function of time after start of treatment for all 5 tumour models. For each of the 4 different treatment arms growth time is presented as the time for tumours to reach the 2-fold (GDV2) and 5-fold (GDV5) of the starting volume. The application of BIBW 2992 alone leads to a significant prolongation of tumour growth time in all tumour models with considerable intertumoural heterogeneity. While for the A7 tumour model only a slight prolongation of tumour growth can be seen, a distinct effect was observed for the FaDu-, A431-, UT-SCC-14 and UT-SCC-15 tumour models. A complete regression of all tumours during continuous application of BIBW 2992 alone without recurrence seen during treatment time (mean 73 days) was determined in the UT-SCC-14, A431 and UT-SCC-15 tumours. During fractionated irradiation (15 fractions within 15 days, total dose 30 Gy) a significant prolongation of tumour growth by BIBW 2992 could be observed for the A7, A431 and UT-SCC-15 tumour models where as this effect was not evident in FaDu and UT-SCC-14 tumours (Table 1). The larger growth inhibiting effect of BIBW 2992 alone compared to the combination with fractionated irradiation can be seen as a consequence of the shorter administration within the combined treatment schedule.