**3.** *In Vivo* **experiments**

402 Advances in the Biology, Imaging and Therapies for Glioblastoma

(Short et al., 1994a, 2001). Cell survival was calculated by DMIPS cell assay after 15 fractions of 0.4 Gy, given three times a day and compared to the same total dose given as once-daily 1.2 Gy. The low-doses were administered at 4-hours intervals (09.00 – 13.00 – 17.00 hours) each day for 5 consecutive days, and the single dose of 1.2 Gy was also given for 5 consecutive days (Short et al., 1994a, 2001). The repeated low-doses produced a significantly increased tumor cell kill; cell survival after three consecutive 0.4 Gy fractions was lower than after the same total dose given as a single fraction (1.2 Gy), the difference was significant (p<0.0002) (Short et al., 1994a, 2001). Cell survival after 2 Gy single doses was not different to that obtained after three consecutive 0.4 Gy fractions (Short et al., 1994a, 2001).Two other human glioblastoma cell lines, A7 – U87, were also tested: the lowest cell survival occurred with doses administered at 4 and 6 hours intervals for A7 and at 1 and 5 hours intervals for U87 (Short et al., 1994a, 2001). The cell line U373 (obtained from a human astrocytoma grade III) did not express HRS phenomenon, repeated low-doses did not enhance cell killing (Short et al., 1994a, 2001). The conclusions of this work were that multiple low-doses (< 1 Gy) per day spaced at appropriate intervals (4 hours) could increase cell killing by the enhancing he HRS phenomenon (Short et al., 1994a, 2001). The authors termed this multiple low-doses per fraction per day as an

G5 CL35 G152 MRC5

Beauchesne et al also tested the cumulative effect of low radiation doses on cell survival on the following human glioblastoma cell lines; G5 – CL35 – G152 (Beauchesne et al., 2003; Pedeux et al., 2003). Three fractions of 0.8 Gy spaced at 4 hour intervals were compared to a biologically equivalent single dose of 2 Gy. Irradiations were given for 2 consecutive days. A marked increase in cell killing was reported with the ultrafractionated regimen (repeated low-doses) in the G5 and G152 cell lines, but not in the CL35 cell line (Fig. 3) (Beauchesne et al., 2003; Pedeux et al., 2003). The experiments were repeated with a linear accelerator used daily for clinical therapies for patients. G5 and CL35 cell lines were exposed to 0.8 Gy, three times per day, spaced at 4 hour intervals for 2 consecutive days and to 2.4 Gy once a day for

Fig. 3. Survival of human glioma cells following repeated irradiations. G5, CL35, G152 or MRC5 cells were exposed to 0.8 Gy 3 times/day spaced by 4 hr for 2 consecutive days or to 2 Gy once/day for 2 consecutive days. Cell survival was assessed by a clonogenic assay.

0,8 2

"ultrafractionated regimen" (Short et al., 1994a, 2001).

0

10

20

30

SF %

40

50

The first study which tested ultrafractionated irradiation on an animal model was reported by Beck-Bornholdt; the rat rhabdomyosarcoma R1H was irradiated with 126 fractions over 6 weeks. Top-up irradiations were not given (different doses per fraction between 0.43 and 0.71 Gy were applied) (Beck-Bornholdt et al., 1989). The results were compared to "historical control", and the authors demonstrated that the ultrafractionated regimen was slight more effective than the conventional approach (Beck-Bornholdt et al., 1989).

With a view to demonstrating a potential therapeutic benefit of the ultrafractionated irradiation schedule for malignant glioma patients, Beauchesne et al tested the fractionated low-dose irradiation in a glioma animal model, previously developed by the same team, on the G152 cell line (Beauchesne et al., 2003; Pedeux et al., 2003). Briefly the model was developed as follows; G152 malignant glioma cells (2 x106) suspended in 0.1 ml of PBS were subcutaneously injected into the inter-scapular region of 4-week-old mice (female nude mice, Swiss *nu/nu*). Drinking water was supplemented with estrone (0.1 ml/100 ml of water) until death of the animal. Two perpendicular diameters (D1 and D2) of the tumors were measured once a week and tumor volume calculated from the following equation: (D1 + D2/2)3 X (/6) (Beauchesne et al., 2003; Pedeux et al., 2003). G152 xenograft tumors were grown for 17 days, and the mice were then exposed to either 0.8 Gy per fraction (3 times per day, spaced at 4 hour intervals, 4 days per week, for 2 consecutive weeks) or to a single dose of 2 Gy (once per day, 4 days per week, for 2 consecutive weeks). Another arm of tumorbearing mice were not treated. The ultrafractionated irradiation was delivered by a clinical linear accelerator, with the mice immobilized in plastic tubes, and only the tumor exposed to the irradiation (Beauchesne et al., 2003; Pedeux et al., 2003).

Tumors grew faster in the untreated mice with an average tumor volume at week 12 of 1223 mm3. As expected, radiation therapy had a therapeutic effect on the tumor growth resulting in an inhibition of tumor growth of 80-90 % (Beauchesne et al., 2003; Pedeux et al., 2003). At week 12, tumor volume of the mice in the ultrafractionated arm (repeated low-doses) was half that of the mice in the standard treatment arm (single dose, each day) representing a highly significant difference (p=0.0022) (Beauchesne et al., 2003; Pedeux et al., 2003). A second experiment gave similar results with neuropathology analysis revealed that the grafted tumor had the same characteristics as the initial human primary glioma tumor from which the G152 was obtained.

To further assert the therapeutic efficiency of ultrafractionated regimen, a third experiment was performed to compare the irradiation regimens for the same total doses (Beauchesne et al., 2003; Pedeux et al., 2003). Seventeen days after grafting, the mice were exposed to either 0.8 Gy, 3 times/day spaced at 4 hour intervals 5 days/week for 2 consecutive weeks (total dose = 24 Gy) or to 2.4 Gy once/day 5 days/week for 2 consecutive weeks (total dose = 24 Gy). Another group of mice was left untreated. Tumor size was measured once a week. As previously demonstrated, the ultrafractionated regimen led to a dramatic inhibition of tumor growth, and in the group of mice irradiated with fractions of 2.4 Gy, the tumor growth was not very different from mice irradiated with 2 Gy per fractions (Fig. 4)

Ultrafractionated Radiation Therapy (3 daily doses of 0.75 Gy) -

The ultrafractionated radiation therapy regimen

unresectable glioblastoma (Beauchesne et al., 2010).

glioblastoma patients (Beauchesne et al., 2010).

(Krause et al., 2003).

**4. Clinical trials** 

**4.1 The population** 

**4.2 Treatment** 

and reproducibility.

**4.3 Patient evaluation** 

A New and Promising Radiotherapy Schedule for Glioblastoma Patients 405

authors were unable to demonstrate a therapeutic benefit of the ultrafractionated regimen; growth delay in the ultrafractionated arm was significantly shorter than after conventional treatment (Krause et al., 2003).The top-up TCD50 values were 28.3 Gy for conventional irradiation and 40 Gy for the ultrafractionated regimen (p=0.047) (Krause et al., 2003). The use of a local irradiator with a dose rate 0.2 – 0.4 Gy/min could alter the molecular mechanisms and perhaps modify the mechanisms responsible for the HRS phenomenon

To translate these *in vitro* and *in vivo* observations into a clinical setting, Beauchesne et al initiated a phase I/II clinical trial using an ultrafractionated radiation therapy protocol (3 times 0.75 Gy for a total of 67.5 Gy) (Beauchesne et al., 2010). The main purpose of this study was to assess the toxicity of the ultrafractionated regimen. This protocol was initiated before concomitant radio-chemotherapy became standard of care. For this pilot trial, the authors purposely selected patients with unfavourable clinical prognostic factors: newly

This phase I/II study was conducted in seven French centres. Patients (> 18 years and who were able to give informed consent) with newly diagnosed, supratentorial, unresectable but histologically confirmed glioblastoma (astrocytoma grade IV according to the WHO classification), with a WHO performance status of 0-2 were eligible (Beauchesne et al., 2010). Patients were included based on local pathology. Patients with an estimated survival of less than 3 months, who had undergone partial or complete tumor resection, or who had previously received prior radiation therapy were not eligible. The primary end-points of the study were the treatment-related toxicity and tolerability (Beauchesne et al., 2010). Secondary end-points were progression-free survival (PFS) and overall survival (OS) of

The radiation therapy regimen consisted of ultrafractionated focal irradiation with three daily doses of 0.75 Gy delivered at least four hours apart five days a week (Monday through Friday), for 6-7 consecutive weeks. A total dose of 67.5 Gy was delivered in 90 fractions (Beauchesne et al., 2010). Irradiation was delivered to the gross tumor volume with a 2.5 cm margin for the clinical target volume. Radiation therapy was planned with dedicated computed tomography (CT) or magnetic resonance imaging (MRI), and three-dimensional planning systems; conformal ultrafractionated radiotherapy was delivered with linear accelerators with a nominal energy of 6 MeV or more (Beauchesne et al., 2010). The patients were treated with thermoplastic immobilization masks to ensure adequate immobilization

The patients were assessed weekly for tolerability and toxicity during the radiation therapy. The baseline examination included cranial MRI (with and without contrast), physical and

(Beauchesne et al., 2003; Pedeux et al., 2003). These experiments show that ultrafractionated irradiation provides a marked benefit compared to a more classical irradiation regimen. It is worth noting that the use of a clinical linear accelerator is more feasible and the ultrafractionated regimen could thus be suitable for clinical treatment (Beauchesne et al., 2003; Pedeux et al., 2003).

Fig. 4. Inhibition of glioma tumor growth following repeated irradiation with low doses. G152 glioma cells injected into the interscapular region of 4-week-old female nude mice (Swiss *nu/nu*). Seventeen days after grafting, mice were exposed either to 0.8 Gy 3 times/day spaced by 4 hr 5 days/week for 2 consecutive weeks or to 2.4 Gy once/day 5 days/week for 2 consecutive weeks. Tumor size was measured once a week.

Krause et al tested the ultrafractionated regimen in an animal model grafted with cells derived from the A7 human cell line which expressed the HRS phenomenon (Krause et al., 2003). Cryoprereserved tumor tissue was transplanted subcutaneously onto the backs of ve mice. The tumor with the median volume doubling time was transplanted onto the back of another eight to 12 mice. 1–2 mm pieces of tissue from the median tumor were then transplanted subcutaneously into the right hind leg of the experimental animals (Krause et al., 2003). Irradiation protocols were started daily when the tumours reached a mean diameter of 5 mm, corresponding to a volume of 57 mm3; the ultrafractionated regimen consisted of 26 fractions over 6 weeks (0.4 Gy per fraction, 3 fractions per day, 21 fractions per week, spaced at 6 hour intervals), and conventional treatment consisted of 30 fractions over 6 weeks (1.68 Gy per fraction, once daily, 5 fractions per week) (Krause et al., 2003). A local irradiator was used. Endpoints were tumor growth delay and local tumor control 180 days after the end of the treatment, and for the first 60 days after the end of irradiation. The tumors were measured twice weekly and once weekly thereafter (Krause et al., 2003). The authors were unable to demonstrate a therapeutic benefit of the ultrafractionated regimen; growth delay in the ultrafractionated arm was significantly shorter than after conventional treatment (Krause et al., 2003).The top-up TCD50 values were 28.3 Gy for conventional irradiation and 40 Gy for the ultrafractionated regimen (p=0.047) (Krause et al., 2003). The use of a local irradiator with a dose rate 0.2 – 0.4 Gy/min could alter the molecular mechanisms and perhaps modify the mechanisms responsible for the HRS phenomenon (Krause et al., 2003).
