**4. Clinical studies of combining radiation and immune therapy**

The expression profiles of CD4(+) and CD8(+) T cells and Treg from patients with newly diagnosed *glioblastoma multiforme* are quite different when compared with normal healthy volunteers (Learn, et al. 2006). But how various absorbed dose or various fractionation pattern or methods of radiation delivery can affect T-cell populations and alternative regulatory molecules in glioma patients is still under debate (Chiba, et al. 2010; Teitz-Tennenbaum, et al. 2008; Verastegui, et al. 2003).

#### **4.1 Effects of concomitant temozolomide and radiation therapies on WT1-specific T-cells in malignant glioma**

Like many other solid tumours, glioma have been found to express a protein characteristic for Wilms' tumour 1 (WT1) (Hashiba, et al. 2007). A peptide based immunotherapy targeting the WT1 gene has successfully been used in patients with recurrent glioma. The clinical response indicates that CD8(+) cytotoxic T lymphocytes (CTLs) are the main effectors of this WT1 vaccination (Oka, et al. 2004). A phase II clinical trial of the WT1 vaccination for patients with recurrent malignant glioma resulted in a partial response rate of 9.5% but none complete response. The median length of period with progression-free survival was 20 weeks (Izumoto, et al. 2008).

In planning for a clinical trial of WT1 vaccination involving patients with newly diagnosed malignant glioma, it is also aimed to combine concurrent radiation /TMZ therapy with WT1 immunotherapy. The critical question is, however, if the depletion of lymphocytes caused by the current standard radiation/TMZ treatment is a drawback for a combination with WT1 immunotherapy. Therefore a clinical study was performed in order to determine how the concomitant radiation/TMZ therapy affects the WT1-specific T-cells and other T-cells in terms of their frequencies and total numbers. This study concluded that, even after the decrease of the absolute numbers of lymphocytes, the fraction of WT1 specific T-cells was stable. They concluded that it may the possible to apply WT1 immunotherapy after the end of 6 weeks of radiation/TMZ therapy (Chiba, et al. 2010).

In another clinical study of 8 patients with primary glioma it was found that concomitant radiation/TMZ therapy integrated with autologous dendritic cell-based immunotherapy was feasible and well tolerated. The median progression-free survival (PFS) was 75% and at 6 months and 50% at 18 months. The median time of survival for all patients is 24 months. One patient was still free from progression or recurrence at 34 months (Ardon, et al. 2010).

#### **4.2 Treatment recurrent malignant glioma with hypo-fractionated radiotherapy combined with immune therapy**

A single fraction of high dose radiation therapy has been demonstrated to dramatically increase the priming of T-cell in draining lymphoid tissues, which increased the action of the CD8(+) T cells and lead to reduction and eradication of the primary tumour or distant

Radiation Immune Modulation Therapy of Glioma 377

Although the total lymphocyte count decrease as a consequence of the current radiation/temozolomide therapy, it seems not affect the frequency of antigen specific Tcells, which suggest that combination with immunotherapy might be successful (Ardon, et

This chapter is dedicated to emeritus professor Leif G. Salford who spent his career as neuro-surgeon to fight against the "*guerrilla cells*" of glioma. He initiated the Brain Immuno Gene Tumour Therapy project "BRIGTT" with support of Märit and Hans Rausing Charitable Foundation. Berta Kamprad's foundation of cancer and the Faculty of Medicine at Lund University are gratefully acknowledged for their support in publishing

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al. 2010; Chiba, et al. 2010).

**6. Acknowledgement** 

this chapter.

**7. References** 

metastasis. This immune response, however, is abrogated by conventional fractionated RT or adjuvant chemotherapy (Lee, et al. 2009). So far only preclinical studies of hypofractionated radiation therapy in combination with immune therapy have been performed. The results are however encouraging and clinical trials using this therapeutic regime is urgently needed for both primary and recurrent glioma (Newcomb, et al. 2006; B. R. R. Persson, et al. 2002; B. R. R. Persson, et al. 2010; B. R. R. Persson, et al. 2008).

Henke et al. (2010) found that retreatment of recurrent high-grade glioma with hypofractionated radiation therapy with 20 Gy given over 1 week seems to be feasible even after a previous complete course of radiotherapy (Henke, et al. 2009). Thus it should be feasible to consider hypo-fractionated radiotherapy with about 8 Gy in one or two fractions to recurrent glioma in combination with immune therapy.

#### **4.3 Treatment of newly diagnosed glioma with fractionated radiotherapy combined with vaccination therapy**

An autologous formalin-fixed tumor vaccine (AFTV) has been prepared from formalinfixed and/or paraffin-embedded glioma tumor tissue obtained upon surgery and premixed with original adjuvant materials. In a clinical pilot study, AFTV inoculations of 12 patients took place at least 4 weeks after prior primary conventional glioma treatments were concluded. Of these 12 patients, four responded to the AFTV therapy: one showed a complete response, one showed a partial response, two showed minor responses, and one had stabilization of disease. The median survival period was about 11 months from the initiation of the AFTV treatment. But three of these patients survived for 20 months or more after AFTV inoculation (Ishikawa, et al. 2007). In a subsequent phase I/IIa clinical trial, the AFTV was inoculated in 24 patients with newly diagnosed glioblastoma multiforme, in combination with conventional fractionated radiotherapy. The treatment protocol in that study included aggressive tumor resection, fractionated radiotherapy, 2 Gy per fraction, up to a total dose of 60 Gy, and 3 concomitant courses of AFTV administered with an interval of one week during the last 3 weeks of irradiation. The median duration of overall survival was 21.4 months (95% CI 13.8–31.3 months). The actuarial 2-year survival rate was 40%. These results demonstrate that vaccine treatment in combination with fractionated radiotherapy may be effective in patients with newly diagnosed glioblastoma (Muragaki, et al. 2011). Since the previous pilot study with AFTV therapy only, also has shown a good response, the outcome of the phase I/IIa clinical trial might have been even better if it has been combined with hypo-fractionated radiation therapy as described in the previous paragraph 4.3.

#### **5. Summary and conclusion**

Many pre-clinical models have proven that one or two radiotherapy fractions with a total absorbed dose in the range of 5 - 16 Gy in combination with immune therapy result in enhanced therapeutic response to glioma. This finding opens for the possibility of clinical testing of new challenging therapeutic regimes for glioma, based on a combination of immune-therapy and hypo-fractionated radiotherapy. A regime of one or two radiation sessions with a total radiation target dose in the order of 8 Gy in combination with clinically proven immunotherapy seem so be adequate (De Vleeschouwer, et al. 2008; Gulley, et al. 2005; J. Nemunaitis, et al. 2006a; J. J. Nemunaitis, et al. 2006b; Newcomb, et al. 2010; B. R. R. Persson, et al. 2010; Salford, et al. 2006; Salford, et al. 2004).

Although the total lymphocyte count decrease as a consequence of the current radiation/temozolomide therapy, it seems not affect the frequency of antigen specific Tcells, which suggest that combination with immunotherapy might be successful (Ardon, et al. 2010; Chiba, et al. 2010).
